Fhm a novel member of the TNF ligand supergene family: materials and methods for interaction modulators

ABSTRACT

The present invention provides a purified polynucleotide encoding a novel polypeptide, designated Fhm, which belongs to the TNF gene superfamily; to purified Fhm polypeptide molecules; to antibodies that bind Fhm; to materials comprising such molecules; and to methods of using such molecules.

RELATED APPLICATIONS

[0001] This application claims priority, under U.S.C. § 119, from U.S.provisional patent application Ser. No. 60/147,294 filed Aug. 4, 1999.

FIELD OF THE INVENTION

[0002] The present invention is in the field of recombinant genetics. Inparticular, the present invention relates to a novel receptor ligand,designated Fhm, belonging to the TNF ligand supergene family and nucleicacid molecules encoding the same. The invention also relates to vectors,host cells, anti-Fhm antibodies, and recombinant methods for producingFhm polypeptides. The invention also relates to the use of therecombinant Fhm polypeptide to identify putative bindingpartners/receptors. In addition, provided for are methods and reagentsfor the diagnosis of diseases associated with abnormal Fhm or abnormalexpression of its putative receptor, and methods and pharmaceuticalcompositions for the treatment of diseases associated with abnormal Fhmor abnormal expression of Fhm and/or its receptor. The invention alsodiscloses pharmaceutical compositions for use in the treatment of thesediseases.

BACKGROUND OF THE INVENTION

[0003] Technical advances in the identification, cloning, expression andmanipulation of nucleic acid molecules have greatly accelerated thediscovery of novel therapeutics based upon deciphering the human genome.Rapid nucleic acid sequencing techniques can now generate sequenceinformation at unprecedented rates, and coupled with computationalanalyses, allow the assembly of overlapping sequences into entire genomeand the identification of polypeptide-encoding regions. Comparison of apredicted amino acid sequence against a database compilation of knownamino acid sequences can allow one to determine the extent of homologyto previously identified sequence and/or structure landmarks. Cloningand expression of a polypeptide-encoding region of a nucleic acidmolecule provides a polypeptide product for structural and functionalanalysis. Manipulation of nucleic acid molecules and encodedpolypeptides to produce variants and derivatives thereof may conferadvantageous properties on a product for use as a therapeutic.

[0004] However, in spite of the significant technical advances in genomeresearch over the past decade, the potential for development of noveltherapeutics based on the human genome is still largely unrealized.While a number of genes encoding potentially beneficial proteintherapeutics, or those encoding polypeptides which may act as “targets”for therapeutic molecules, have been identified using recombinant DNAtechnology, the structure and function of a vast number of genes in thegenome of mammals are yet unknown.

[0005] Identification and Characterization of TNF-Family of Ligands andReceptors

[0006] Tumor necrosis factor (TNF) was first identified in the serum ofmice and rabbits which had been infected with bacillus of Calmette andGuerin(BCG) and which had been injected with endotoxin. TNF activity inthe serum of these animals was recognized on the basis of its cytotoxicand anti-tumor activities. This TNF activity, referred to as TNF-α, isproduced particularly by activated monocytes and macrophages, and hasbeen implicated in normal growth processes as well as in a variety ofdiseases.

[0007] Following the discovery of TNF-α, independent research led to theidentification of another cytokine associated with inflammatoryresponses lymphotoxin-α (LT-α), which was shown to be producedexclusively by lymphocytes. LT-α was subsequently shown to be 30%homologous with TNF-α, and was renamed TNF-β. It is now clear that TNF-αand TNF-β are members of a gene family that includes yet another membertermed LT-β (Browning et al., Cell 72:847-856, 1993). The three genesare tightly linked within the MHC complex and show similar organization.Moreover, the biologically active forms of TNF-α and TNF-β arehomotrimers and share many of the same biological activities includingcompeting for the same cell-surface receptors (Aganval et al., Nature318:665-667, 1985). Two distinct but structurally homologous receptorshave been identified, and each has been shown to bind both the ligandsand mediate their effects.

[0008] However, it has been recognized that TNFs are only representativemembers of the rapidly expanding supergene familiy that includes TNF-α,TNF-β/lymphotoxin-α (LT-α), lymphotoxin-β (LT-β), FasL, CD40L, CD30L,CD27L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), RANKL,GITRL and TNF-2. See generally Orlinick et al., Cell Signal, 10(8):543-551 (1998). The distinctive but overlapping cellular responsesinduced by members of the TNF family of ligands following theirinteraction(s) with their cognate cell-surface receptors result inclearly defined developmental and regulatory changes in cells of thelymphoid, hematopoietic, and other lineages. For example, the TNF familyof ligands are involved in growth regulation and differentiation ofcells which are involved in inflamation, immune processes andhematopoiesis (Bayert, R. and Fiers, W., Tumor Necrosis Factor andLymphokines in: Cytokines eds. Anthony Mire-Sluis and Robin Thorpe,Academic Press San Diego Calif., 1998). The TNF family of ligandsactivates the immune defenses against parasites, and act directly orindirectly as mediators in immune reactions and inflammatory processes.However, administration of TNF and/or other members of the TNF familycan also be accompanied by harmful phenomena such as shock and tissuedamage (Bayert, R. and Fiers, W., supra). The main physiological role ofthe TNF family of ligands is the activation of first-line reaction of anorganism to microbial, parasitic, viral infections, or to mechanicalstress and cancer. For example, TNF-related apoptosis-inducing ligand(TRAIL) has been demonstrated to induce apoptosis of a number ofdifferent types of cancer cells as well as virally infected cells.

[0009] Furthermore, a number of observations have also led to theconclusion that the TNF family of ligands are also involved in a varietyof pathological conditions including cachexia, toxic shock syndrome,inflammatory diseases such as rheumatoid and osteoarthritis, and inlethality resulting from graft-versus-host reaction (GVHR) rapidnecrosis of tumors, apoptosis, immunostimulation and resistance toparasites and viruses. (Bayert, R. and Fiers, W., supra).

[0010] Like other cytokines, the members of the TNF family of ligandsact via specific cell-surface receptors. The receptors, with twoexceptions, are type 1 membrane-associated proteins. Sequence homologyamongst them is almost entirely confined to their extracellular domains.For example, two TNF receptors have been cloned which differ in size andin binding affinity (Bayert, R. and Fiers, W., supra). Both receptorsbind TNF-α and TNF-β and are membrane associated proteins. The tworeceptors consist of extracellular domains which bind TNF (and arehomologous for 28%), single transmembrane domains, and intracellulardomains which are totally different from each other and which do notcontain any recognizable structural motifs that have been associatedwith any particular function. Based on similarities in theirextracellular domains, these receptors belong to a receptor genesuperfamily that includes the low-affinity nerve growth factor (NGF)receptor, the Fas antigen, the human B-lymphocyte activation moleculeCD40, CD27, 4-1BB, PV-T2, CD30, TNFR-RP, TRAIL-R, PV-A53R, RANK, GITR,and the OX40 antigen found on activated T-cells (Smith et al., Cell,76(6):959-962, 1994; Baker and Reddy, Oncogene, 12(1):1-9, 1996).

[0011] In addition to the membrane associated receptor moleculesdescribed above, a number the receptors belonging to the TNF-receptorsupergene family exist as soluble ligand binding proteins. Many of thesoluble forms of the transmembrane receptors were subsequentlyidentified as containing only the extracellular ligand binding domain(s)of the receptors. For example, a soluble form of TNF receptor has beenfound in urine and serum (See U.S. Pat. No. 5,843,789 and Nophar et al.,EMBOJ., 9(10):3269-3278, 1990), and has been shown to arise byproteolytic cleavage of cell surface TNF-receptors (Wallach et al.,Agents Actions Suppl., 35:51-57 1991). These soluble forms of receptormolecules have been implicated in the modulation of TNF activity by notonly interfering with TNF binding to its receptor, but also bystabilizing the TNF structure and preserving its activity, thusprolonging some of its effects (Aderka et al, Cytokine & Growth FactorReviews, 7(3):231-240, 1996).

[0012] The activity of members of the TNF family of ligands is tightlyregulated at the levels of secretion and receptor expression. Additionalregulatory mechanisms are provided by action of specific inhibitoryproteins present on cell surfaces and in biological fluids. While someof these inhibitory proteins have been identified as soluble forms ofreceptor molecules, the identity of many of these cytokine regulatoryproteins are as yet unknown. However, abnormalities in the production ofthese substances might contribute to the pathophysiology of a variety ofdiseases including immune and neoplastic diseases. Besides their role inregulating cytokine activity in vivo, these regulatory molecules holdsignificant potential for therapeutic use as very specificinhibitors/anti-cytokine agents, and as indicators in diagnosis andassessment of immune function and growth parameters in a variety ofautoimmune and malignant diseases.

[0013] Because of the important role of the TNF family of ligands (andtheir receptors) in health and disease, a need exists to identify,isolate, and characterize additional members of the family, for use indiagnosing and treating disease and pathological conditions.

SUMMARY OF THE INVENTION

[0014] The present invention relates to a novel serine/threonine kinasefamily and uses thereof. More specifically, the present inventionrelates to novel Fhm nucleic acid molecules and encoded polypeptides,and uses thereof.

[0015] The invention provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

[0016] (a) the nucleotide sequence set forth in SEQ ID NO: 3;

[0017] (b) a nucleotide sequence encoding the polypeptide set forth inSEQ ID NO: 4;

[0018] (c) a nucleotide sequence which hybridizes under moderately orhighly stringent conditions to the complement of (a) or (b), wherein theencoded polypeptide has an activity of the polypeptide set forth in SEQID NO: 3; and

[0019] (d) a nucleotide sequence complementary to any of (a) through(c).

[0020] The invention also provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

[0021] (a) a nucleotide sequence encoding a polypeptide that is at leastabout 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to thepolypeptide set forth in 4, wherein the polypeptide has an activity ofthe encoded polypeptide set forth in SEQ ID NO: 4 as determined using acomputer program selected from the group consisting of GAP, BLASTP,BLASTN, FASTA, BLASTA, BLASTX, BestFit, and the Smith-Watermanalgorithm;

[0022] (b) a nucleotide sequence encoding an allelic variant or splicevariant of the nucleotide sequence set forth in SEQ ID NO: 3, whereinthe encoded polypeptide has an activity of the polypeptide set forth inSEQ ID NO: 4;

[0023] (c) a nucleotide sequence of SEQ ID NO: 3, (a), or (b) encoding apolypeptide fragment of at least about 25 amino acid residues, whereinthe polypeptide has an activity of the polypeptide set forth in SEQ IDNO: 4;

[0024] (d) a nucleotide sequence encoding a polypeptide that has asubstitution wherein the encoded polypeptide has an activity of thepolypeptide set forth in SEQ ID NO: 4;

[0025] (e) a nucleotide sequence of SEQ ID NO: 3, or (a)-(d) comprisinga fragment of at least about 16 nucleotides;

[0026] (f) a nucleotide sequence which hybridizes under moderately orhighly stringent conditions to the complement of any of (a)-(e), whereinthe encoded polypeptide has an activity of the polypeptide set forth inSEQ ID NO:4; and

[0027] (g) a nucleotide sequence complementary to any of (a)-(e).

[0028] The invention further provides for an isolated nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of:

[0029] (a) a nucleotide sequence encoding a polypeptide set forth in SEQID NO: 4 with at least one conservative amino acid substitution, whereinthe encoded polypeptide has an activity of the polypeptide set forth inSEQ ID NO: 4;

[0030] (b) a nucleotide sequence encoding a polypeptide set forth in SEQID NO: 4 with at least one amino acid insertion, wherein the encodedpolypeptide has an activity of the polypeptide set forth in SEQ ID NO:4;

[0031] (c) a nucleotide sequence encoding a polypeptide set forth in SEQID NO: 4 with at least one amino acid deletion, wherein the encodedpolypeptide has an activity of the polypeptide set forth in SEQ ID NO:4;

[0032] (d) a nucleotide sequence encoding a polypeptide set forth in SEQID NO: 4 which has a C- and/or N- terminal truncation, wherein theencoded polypeptide has an activity of the polypeptide set forth in SEQID NO: 4;

[0033] (e) a nucleotide sequence encoding a polypeptide set forth in SEQID NO: 4 with at least one modification selected from the groupconsisting of amino acid substitutions, amino acid insertions, aminoacid deletions, C-terminal truncation, and N-terminal truncation,wherein the polypeptide has an activity of the encoded polypeptide setforth in SEQ ID NO: 4;

[0034] (f) a nucleotide sequence of (a)-(e) comprising a fragment of atleast about 16 nucleotides;

[0035] (g) a nucleotide sequence which hybridizes under moderately orhighly stringent conditions to the complement of any of (a)-(f), whereinthe encoded polypeptide has an activity of the polypeptide set forth inSEQ ID NO: 4; and

[0036] (h) a nucleotide sequence complementary to any of (a)-(e).

[0037] The invention also provides for an isolated polypeptidecomprising the amino acid sequence selected from the group consistingof:

[0038] (a) the mature amino acid sequence set forth in SEQ ID NO: 4comprising a mature amino terminus at residue 1, and optionally furthercomprising an amino-terminal methionine;

[0039] (b) an amino acid sequence for an ortholog of SEQ ID NO: 4,wherein the polypeptide has an activity of the polypeptide set forth inSEQ ID NO: 4;

[0040] (c) an amino acid sequence that is at least about 70, 75, 80, 85,90, 95, 96, 97, 98, or 99 percent identical to the amino acid sequenceof SEQ ID NO: 4, wherein the polypeptide has an activity of thepolypeptide set forth in SEQ ID NO: 4 as determined using a computerprogram selected from the group consisting of GAP, BLASTP, BLASTN,FASTA, BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm;

[0041] (d) a fragment of the amino acid sequence set forth in SEQ ID NO:4 comprising at least about 25 amino acid residues, wherein thepolypeptide has an activity of the polypeptide set forth in SEQ ID NO:4;

[0042] (e) an amino acid sequence for an allelic variant or splicevariant of either the amino acid sequence set forth in SEQ ID NO: 4, orat least one of (a)-(c) wherein the polypeptide has an activity of thepolypeptide set forth in SEQ ID NO: 4.

[0043] The invention further provides for an isolated polypeptidecomprising the amino acid sequence selected from the group consistingof:

[0044] (a) the amino acid sequence set forth in SEQ ID NO: 4 with atleast one conservative amino acid substitution, wherein the polypeptidehas an activity of the polypeptide set forth in SEQ ID NO: 4;

[0045] (b) the amino acid sequence set forth in SEQ ID NO: 4 with atleast one amino acid insertion, wherein the polypeptide has an activityof the polypeptide set forth in SEQ ID NO: 4;

[0046] (c) the amino acid sequence set forth in SEQ ID NO: 4 with atleast one amino acid deletion, wherein the polypeptide has an activityof the polypeptide set forth in SEQ ID NO: 4;

[0047] (d) the amino acid sequence set forth in SEQ ID NO: 4 which has aC- and/or N-terminal truncation, wherein the polypeptide has an activityof the polypeptide set forth in SEQ ID NO: 4; and

[0048] (e) the amino acid sequence set forth in SEQ ID NO: 4, with atleast one modification selected from the group consisting of amino acidsubstitutions, amino acid insertions, amino acid deletions, C-terminaltruncation, and N-terminal truncation, wherein the polypeptide has anactivity of the polypeptide set forth in SEQ ID NO: 4.

[0049] Also provided are fusion polypeptides comprising the polypeptidesequences of (a)-(e) above of the preceding paragraphs.

[0050] The present invention also provides for an expression vectorcomprising the isolated nucleic acid molecules set forth herein,recombinant host cells comprising recombinant nucleic acid molecules setforth herein, and a method of producing a Fhm polypeptide comprisingculturing the host cells and optionally isolating the polypeptide soproduced.

[0051] A transgenic non-human animal comprising a nucleic acid moleculeencoding a Fhm polypeptide is also encompassed by the invention. The Fhmnucleic acid molecules are introduced into the animal in a manner thatallows expression and increased levels of the Fhm polypeptide, which mayinclude increased circulating levels. The transgenic non-human animal ispreferably a mammal.

[0052] Also provided are derivatives of the Fhm polypeptides of thepresent invention.

[0053] Additionally provided are selective binding agents such asantibodies and peptides capable of specifically binding the Fhmpolypeptides of the invention. Such antibodies and peptides may beagonistic or antagonistic.

[0054] Pharmaceutical compositions comprising the nucleotides,polypeptides, or selective binding agents of the present invention andone or more pharmaceutically acceptable formulation agents are alsoencompassed by the invention. The pharmaceutical compositions are usedto provide therapeutically effective amounts of the nucleotides orpolypeptides of the present invention. The invention is also directed tomethods of using the polypeptides, nucleic acid molecules, and selectivebinding agents. The invention also provides for devices to administer aFhm polypeptide encapsulated in a membrane.

[0055] The Fhm polypeptides and nucleic acid molecules of the presentinvention may be used to treat, prevent, ameliorate, diagnose and/ordetect diseases and disorders, including those recited herein.Expression analysis in biological, cellular or tissue samples suggeststhat Fhm polypeptide may play a role in the diagnosis and/or treatmentof TNF-related diseases including, but not limited to,acquired-immunodeficiency syndrome (AIDS), anemia, autoimmune diseases,cachexia, cancer, cerebral malaria, diabetes mellitus, disseminatedintravascular coagulopathy, erythryoid sick syndrome, hemorrhagic shock,hepatitis, insulin resistance, leprosy, leukemia, lymphoma, meningitis,multiple sclerosis, myocardial ischaemia, obesity, rejection oftransplanted organs, rheumatoid arthritis, septic shock syndrome,stroke, adult respiratory distress syndrome (ARDS), tuberculosis, and anumber of viral diseases. This expression can de detected with adiagnostic agent such as Fhm nucleotide.

[0056] The invention encompasses diagnosing a pathological condition ora susceptibility to a pathological condition in a subject caused by orresulting from abnormal levels of Fhm polypeptide comprising determiningthe presence or amount of expression of the Fhm polypeptide in a sample;and comparing the level of said polypeptide in a biological, tissue orcellular sample from either normal subjects or the subject at an earliertime, wherein susceptibility to a pathological condition is based on thepresence or amount of expression of the polypeptide.

[0057] The present invention also provides a method of assaying testmolecules to identify a test molecule which binds to a Fhm polypeptide.The method comprises contacting a Fhm polypeptide with a test moleculeand to determine the extent of binding of the test molecule to thepolypeptide. The method further comprises determining whether such testmolecules are agonists or antagonists of a Fhm polypeptide. The presentinvention further provides a method of testing the impact of moleculeson the expression of Fhm polypeptide or on the activity of Fhmpolypeptide.

[0058] Methods of regulating expression and modulating (i.e., increasingor decreasing) levels of a Fhm polypeptide are also encompassed by theinvention. One method comprises administering to an animal a nucleicacid molecule encoding a Fhm polypeptide. In another method, a nucleicacid molecule comprising elements that regulate or modulate theexpression of a Fhm polypeptide may be administered. Examples of thesemethods include gene therapy, cell therapy, and anti-sense therapy asfurther described herein.

[0059] Surprisingly, a Fhm polypeptide was highly expressed in a widerange of primary human tumors. Therefore, the present polypeptide, andits useful nucleid acid intermediates, have demonstrated utility indifferentiating transformed cells from the background.

[0060] In another aspect of the present invention, the Fhm polypeptidesmay be used for identifying receptors or binding partners thereof (“Fhmreceptors” or “Fhm binding partners”). Various forms of “expressioncloning” have been extensively used to clone receptors for protein orco-factors. See, for example, Simonsen and Lodish, Trends inPharmacological Sciences, 15: 437-441, 1994, and Tartaglia et al., Cell,83:1263-1271, 1995. The isolation of the Fhm receptor(s) or Fhm bindingpartner(s) is useful for identifying or developing novel agonists andantagonists of the Fhm polypeptide-signaling pathway.

[0061] Such agonists and antagonists include soluble Fhm ligand(s),anti-Fhm selective binding agents (such as Fhm antibodies andderivatives thereof), small molecules, peptides or derivatives thereofcapable of binding Fhm polypeptides, or antisense oligonucleotides, anyof which can be used for potentially treating one or more diseases ordisorders, including those recited herein.

[0062] In certain embodiments, a Fhm polypeptide agonist or antagonistmay be a protein, peptide, carbohydrate, lipid, or small molecularweight molecule which interacts with Fhm polypeptide to regulate itsactivity.

[0063] In another aspect of the present invention, the Fhm polypeptidesmay be used for identifying receptors thereof (“Fhm receptors”). Variousforms of “expression cloning” have been extensively used for cloning toclone receptors for protein ligands. See for example, H. Simonsen and H.F. Lodish, Trends in Pharmacological Sciences, vol. 15,437-44115:437-44, 1994, and Tartaglia et al., Cell, 83:1263-1271, 1995.The isolation of the Fhm receptor(s) is useful for identifying ordeveloping novel agonists and antagonists of the Fhmpolypeptide-signaling pathway. Such agonists and antagonists includesoluble Fhm receptor(s), anti-Fhm receptor selective receptor-selectivebinding agents (such as antibodies and derivatives thereof), smallmolecules, and antisense oligonucleotides, any of which can be used fortreating one or more of the diseases or disorders, including thoserecited herein.

DESCRIPTION OF THE FIGURE

[0064]FIG. 1 presents an alignment of the predicted amino acid sequenceof Fhm polypeptide (SEQ ID NO: 4) is aligned with the correspondingregions of human FasL, mouse FasL, rat FasL, human CD40L, mouse CD40L,mouse OPGL, human OPGL, human TRAIL, mouse TRAIL, human CD30L, humanCD30L, human LyT-β, mouse LyT-β, human TNF-β, mouse TNF-β, human TNF-αand mouse TNF-α. (SEQ ID NOS: 5-21) using the Pileup program (WisconsinGCG Program Package ver. 8.1).

DETAILED DESCRIPTION OF THE INVENTION

[0065] The section headings herein are for organizational purposes onlyand are not to be construed as limiting the subject matter describedtherein.

[0066] Definitions:

[0067] The terms “Fhm gene”, “Fhm nucleic acid molecule”, or “Fhmpolynucleotide” refer to a nucleic acid molecule comprising orconsisting essentially of a nucleotide sequence as set forth in SEQ IDNO: 3, comprising or consisting essentially of a nucleotide sequenceencoding the polypeptide as set forth in SEQ ID NO: 4, or nucleic acidmolecules related thereto. “Related” nucleic acid molecules comprise orconsist essentially of a nucleotide sequence that is about 70 percentidentical to the nucleotide sequence as shown in SEQ ID NO: 3, orcomprise or consist essentially of a nucleotide sequence encoding apolypeptide having an amino acid sequence that is about 70 percentidentical to the amino acid sequence set forth in SEQ ID NO: 4. Inpreferred embodiments, the nucleotide sequences are about 75 percent, orabout 80 percent, or about 85 percent, or about 90 percent, or about 95,96, 97, 98, or 99 percent identical to the nucleotide sequence as shownin SEQ ID NO: 3, or the nucleotide sequences encode a polypeptide thatis about 75 percent, or about 80 percent, or about 85 percent, or about90 percent, or about 95, 96, 97, 98, or 99 percent identical to thepolypeptide sequence as set forth in SEQ ID NO: 4. Related nucleic acidmolecules also include fragments of the above Fhm nucleic acid moleculeswhich are at least about 10 contiguous nucleotides, or about 15, orabout 20, or about 25, or about 50, or about 75, or about 100, orgreater than about 100 contiguous nucleotides. Related nucleic acidmolecules also include fragments of the above Fhm nucleic acid moleculeswhich encode a polypeptide of at least about 25 amino acid residues, orabout 50, or about 75, or about 100, or greater than about 100 aminoacid residues. Related nucleic acid molecules also include a nucleotidesequence encoding a polypeptide comprising or consisting essentially ofa substitution and/or a deletion of one to 251 amino acid residuescompared to the polypeptide in SEQ ID NO: 4. Related Fhm ligand nucleicacid molecules include those molecules which comprise nucleotidesequences which hybridize under moderate or highly stringent conditionsas defined herein with any of the above nucleic acid molecules. Inpreferred embodiments, the related nucleic acid molecules comprisesequences which hybridize under moderate or highly stringent conditionswith the sequence as shown in SEQ ID NO:3, or with a molecule encoding apolypeptide, which polypeptide comprises the amino acid sequence asshown in SEQ ID NO:4, or with a nucleic acid fragment as defined above,or with a nucleic acid fragment encoding a polypeptide as defined above.It is also understood that related nucleic acid molecules includeallelic or splice variants of any of the above nucleic acids, andinclude sequences which are complementary to any of the above nucleotidesequences.

[0068] The term “Fhm polypeptide allelic variant” refers to one ofseveral possible naturally occurring alternate forms of a gene occupyinga given locus on a chromosome of an organism or a population oforganisms.

[0069] the term “Fhm polypeptide splice variant” refers to a nucleicacid molecule, usually RNA, which is generated by alternative processingof intron sequences in an RNA transcript of Fhm polypeptide amino acidsequence set forth in SEQ ID NO: 4.

[0070] The term “expression vector” refers to a vector which is suitablefor use in a host cell and contains nucleic acid sequences which directand/or control the expression of inserted heterologous nucleic acidsequences. Expression includes, but is not limited to, processes such astranscription, translation, and RNA splicing, if introns are present.

[0071] The term “Fhm polypeptide” refers to a polypeptide comprising theamino acid sequence of SEQ ID NO: 4, and related polypeptides. Relatedpolypeptides includes: Fhm allelic variants, Fhm splice variants, Fhmfragments, Fhm derivatives, Fhm-substitution, -deletion, and/orinsertion variants, Fhm fusion polypeptides, and Fhm orthologs. Fhmpolypeptides may be mature polypeptides, as defined herein, and may ormay not have an amino terminal methionine residue, depending on themethod by which they are prepared.

[0072] The term Fhm polypeptide fragment refers to a peptide orpolypeptide that comprises less than the full length amino acid sequenceof a Fhm polypeptide as set forth in SEQ ID NO: 4. Such a fragment mayarise, for example, from a truncation at the amino terminus (with orwithout a leader sequence), α-truncation at the carboxy terminus, and/oran internal deletion of the amino acid sequence (wherein the resultingpolypeptide is at lease 6 amino acids or more in length). Fhm fragmentsmay result from alternative RNA splicing or from in vivo proteaseactivity.

[0073] In preferred embodiments, truncations comprise about 10 aminoacids, or about 20 amino acids, or about 50 amino acids, or about 75amino acids, or about 100 amino acids, or more than about 100 aminoacids. The polypeptide fragments so produced will comprise about 25contiguous amino acids, or about 50 amino acids, or about 75 aminoacids, or about 100 amino acids, or about 150 amino acids, or about 200amino acids. Such Fhm polypeptide fragments may optionally comprise anamino terminal methionine residue. It will be appreciated that suchfragments can be used, for example, to generate antibodies to Fhmpolypeptides.

[0074] The term “Fhm polypeptide variants” refers to Fhm polypeptidescomprising amino acid sequences which contain one or more amino acidsequence substitutions, deletions (such as internal deletions and/or Fhmfragments), and/or additions (such as internal additions and/or Fhmfusion polypeptides) as compared to the Fhm polypeptide amino acidsequence set forth in SEQ ID NO: 4 (with or without leader sequences).Variants may be naturally occurring (e.g., Fhm polypeptide allelicvariants, Fhm polypeptide orthologs or Fhm splice variants) orartificially constructed. Such Fhm-polypeptide variants may be preparedfrom the corresponding nucleic acid molecules encoding said variants,which have a DNA sequence that varies accordingly from the DNA sequencesfor wild type Fhm-receptor polypeptides as set forth in SEQ ID NO: 3.

[0075] The term “Fhm fusion polypeptide” “Fhm fusion polypeptide” refersto a fusion of one or more amino acids (such as a heterologous peptideor polypeptide) at the amino or carboxy terminus of the polypeptide asset forth in SEQ ID NO: 4, Fhm polypeptide allelic variants, Fhmpolypeptide orthologs, Fhm polypeptide splice variants, or Fhmpolypeptide variants having one or more amino acid deletions,substitutions or internal additions as compared to the Fhm polypeptideamino acid sequence set forth in SEQ ID NO: 4.

[0076] The term “Fhm polypeptide derivatives” refers to Fhmpolypeptides, variants, or fragments thereof, that have been chemicallymodified, as for example, by covalent attachment of one or more watersoluble polymers, N-linked or O-linked carbohydrates, sugars,phosphates, and/or other such molecules. Such modifications may beintroduced into the molecule by reacting targeted amino acid residues ofthe purified or crude protein with an organic derivatizing agent that iscapable of reacting with selected side chains or terminal residues. Theresulting covalent derivatives are also useful in programs directed atidentifying residues important for biological activity. The derivativesare modified in a manner that is different from naturally occurring Fhmpolypeptide either in the type or location of the molecules attached tothe polypeptide. Derivatives further include deletion of one or morechemical groups naturally attached to the Fhm polypeptide.

[0077] The terms “biologically active Fhm polypeptides”, “biologicallyactive Fhm polypeptide fragments”, “biologically active Fhm polypeptidevariants”, and “biologically active Fhm polypeptide derivatives” referto Fhm polypeptides having at least one activity characteristic of a Fhmpolypeptide comprising the amino acid sequence of SEQ ID NO: 4, such asthe ability to bind to one or more members of the TNF-receptor supergene (protein) family in biological assays. Immunogenic fragments of Fhmpolypeptides are those capable of inducing in a host animal antibodiesdirected to the Fhm fragment.

[0078] The term “Fhm polypeptide ortholog” refers to a polypeptide fromanother species that corresponds to a Fhm polypeptide amino acidsequence set forth as SEQ ID NO: 4. For example, mouse and human Fhmpolypeptides are considered orthologs of each other.

[0079] The term “mature Fhm polypeptide” refers to a Fhm polypeptidelacking a leader sequence, a mature Fhm polypeptide may also includeother modifications of a polypeptide such as proteolytic processing ofthe amino terminus (with or without a leader sequence) and/or thecarboxy terminus, cleavage of a smaller polypeptide from a largerprecursor, N-linked and/or O-linked glycosylation, and the like.

[0080] The term “antigen” refers to a molecule or a portion of amolecule capable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that each antigen. Anantigen may have one or more epitopes.

[0081] The term “mutein” refers to a mutant protein, polypeptide,variants, analogs or fragments of Fhm polypeptide. Muteins of Fhm may beprepared by deletion, insertion, substitution, point mutation,truncation, addition, transposition, PCR amplification, site-directedmutagenesis or other methods known in the art.

[0082] The terms “effective amount” and “therapeutically effectiveamount” refer to the amount of a Fhm polypeptide (or Fhm antagonist)necessary to support an observable change in the level of one or morebiological activities of one or more members of the TNF-receptor genefamily as set forth above, to bring about a meaningful patient benefit,i.e. treatment, healing, prevention, or amelioration of a condition.When applied to an individual active ingredient, administered alone, theterm refers to that ingredient alone. When applied to a combination, theterm refers to combined amounts of active ingredients that result intherapeutic effect, when administered in combination, serially orsimultaneously. The Fhm polypeptides that have use in practicing thepresent invention may be naturally occurring full length polypeptides,or truncated polypeptides or variant homologs or analogs or derivativesor peptide fragments. Illustrative analogs include those in which one ormore divergent amino acids between two species are substituted with thedivergent amino acid from another species. Divergent amino acids mayalso be substituted with any other amino acid whether it be aconservative or a non-conservative amino acid.

[0083] The term “identity”, as known in the art, refers to arelationship between the sequences of two or more polypeptide moleculesor two or more nucleic acid molecules, as determined by comparing thesequences. In the art, “identity” also means the degree of sequencerelatedness nucleic acid molecules or polypeptides sequences, as thecase may be, as determined by the match between strings of two or morenucleotide or two or more amino acid sequences. “Identity” measures thepercent of identical matches between the smaller of two or moresequences with gap alignments (if any) addressed by particular amathematical model of computer program (i.e., “algorithms”). The term“similarity” is a related concept, but in contrast to “identity”, refersto a measure of similarity which includes both identical matches andconservative substitution matches. -If two polypeptide sequences have,for example, 10/20 identical amino acids, and the remainder are allnon-conservative substitutions, then the percent identity and similaritywould both be 50%. If, in the same example, there are 5 more positionswhere there are conservative substitutions, then the percent identityremains 50%, but the per cent similarity would be 75% (15/20).Therefore, in cases where there are conservative substitutions, thedegree of percent similarity between two polypeptide sequences will behigher than the percent identity between those two sequences.

[0084] The term “isolated nucleic acid molecule” refers to a nucleicacid molecule of the invention that (1) has been separated from at leastabout 50 percent of proteins, lipids, carbohydrates or other materialswith which it is naturally found when total DNA is isolated from thesource cells, (2) is not linked to all or a portion of a polynucleotideto which the “isolated” isolated nucleic acid molecule “molecule” islinked in nature, (3) is operably linked to a polynucleotide which it isnot linked to in nature, or (4) does not occur in nature as part of alarger polynucleotide sequence. Preferably, the isolated nucleic acidmolecule of the present invention is substantially free from any othercontaminating nucleic acid molecule(s) or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use.

[0085] The term “isolated polypeptide” refers to a polypeptide of thepresent invention that (1) has been separated from at least about 50percent of polynucleotides, lipids, carbohydrates or other materialswith which it is naturally found when isolated from the source cell, (2)is not linked (by covalent or noncovalent interaction) to all or aportion of a polypeptide to which the “isolated polypeptide” is linkedin nature, (3) is operably linked (by covalent or noncovalentinteraction) to a polypeptide with which it is not linked in nature, or(4) does not occur in nature. Preferably, the isolated polypeptide issubstantially free from any other contaminating polypeptides or othercontaminants that are found in its natural environment that wouldinterfere with its therapeutic, diagnostic, prophylactic or researchuse.

[0086] The terms “nucleic acid sequence” or “nucleic acid molecule”refer to a DNA or RNA sequence. The term encompassesterms encompassmolecules formed from any of the known base analogs of DNA and RNA suchas, but not limited to 4-4-acetylcytosine, 8-hydroxy-N6-methyladenosine,aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

[0087] The term “naturally occurring” or “native” when used inconnection with biological materials such as nucleic acid molecules,polypeptides, host cells, and the like, refers to materials which arefound in nature and are not manipulated by man. Similarly,“non-naturally occurring” or “non-native” “non-naturally occurring” or“non-native” as used herein refers to a material that is not found innature or that has been structurally modified or synthesized by man.

[0088] The term “operably linked” “operably linked” is used herein torefer to an arrangementa method of flanking sequences wherein theflanking sequences so described are configured or assembled so as toperform their usual function. Thus, a flanking sequence operably linkedto a coding sequence may be capable of effecting the replication,transcription and/or translation of the coding sequence. For example, acoding sequence is operably linked to a promoter when the promoter iscapable of directing transcription of that coding sequence. A flankingsequence need not be contiguous with the coding sequence, so long as itfunctions correctly. Thus, for example, intervening untranslated yettranscribed sequences can be present between a promoter sequence and thecoding sequence, and the promoter sequence can still be considered“operably linked” “operably linked” to the coding sequence.

[0089] The term “pharmaceutically acceptable carrier” or“physiologically acceptable carrier” as used herein refers terms“pharmaceutically acceptable carrier” or “physiologically acceptablecarrier” as used herein refer to one or more formulation materialssuitable for accomplishing or enhancing the delivery of the Fhmpolypeptide, Fhm nucleic acid molecule or Fhm selective binding agent asa pharmaceutical composition.

[0090] The term “selective binding agent” refers to a molecule ormolecules having specificity for an Fhm polypeptide. As used herein, theterms, “specific” and “specificity” refer to the ability of theselective binding agents to bind to human Fhm polypeptides and not tobind to human non-Fhm polypeptides. It will be appreciated, however,that the selective binding agents may also bind orthologs of thepolypeptide as set forth in SEQ ID NO: 4, that is, interspecies versionsthereof, such as mouse and rat polypeptides.

[0091] The term “transduction” is used to refer to the transfer of genesfrom one bacterium to another, usually by a phage. “Transduction” alsorefers to the acquisition and transfer of eukaryotic cellular sequencesby retroviruses.

[0092] The term “transfection” is used to refer to the uptake of foreignor exogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, for example, Graham et al., Virology, 52:456, 1973;Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold SpringHarbor Laboratories, New York, 1989; Davis et al., Basic Methods inMolecular Biology, Elsevier, 1986; and Chu et al., Gene, 13:197, 198 1.Such techniques can be used to introduce one or more exogenous DNAmoieties into suitable host cells.

[0093] The term “transformation” as used herein refers to a change in acell's genetic characteristics, and a cell has been transformed when ithas been modified to contain a new DNA. For example, a cell istransformed where it is genetically modified from its native state.Following transfection or transduction, the transforming DNA mayrecombine with that of the cell by physically integrating into achromosome of the cell, it may be maintained transiently as an episomalelement without being replicated, or I may replicate independently as aplasmid. A cell is considered to have been stably transformed when theDNA is replicated with the division of the cell.

[0094] The term “vector” is used to refer to any molecule (e.g., nucleicacid, plasmid, or virus) used to transfer coding information to a hostcell.

[0095] Relatedness of Nucleic Acid Molecules and/or Polypeptides

[0096] It is understood that related nucleic acid molecules includeallelic or splice variants of the nucleic acid molecule of SEQ ID NO: 3,and include sequences which are complementary to any of the abovenucleotide sequences. Related nucleic acid molecules also include anucleotide sequence encoding a polypeptide comprising or consistingessentially of a substitution, modification, addition and/or a deletionof one or more amino acid residues compared to the polypeptide in SEQ IDNO: 4.

[0097] Fragments include molecules which encode a polypeptide of atleast about 25 amino acid residues, or about 50, or about 75, or about100, or greater than about 100, amino acid residues of the polypeptideof SEQ ID NO: 4.

[0098] In addition, related Fhm nucleic acid molecules include thosemolecules which comprise nucleotide sequences which hybridize undermoderately or highly stringent conditions as defined herein with thefully complementary sequence of the nucleic acid molecule of SEQ ID NO:3, or of a molecule encoding a polypeptide, which polypeptide comprisesthe amino acid sequence as shown in SEQ ID NO: 4, or of a nucleic acidfragment as defined herein, or of a nucleic acid fragment encoding apolypeptide as defined herein. Hybridization probes may be preparedusing the Fhm sequences provided herein to screen cDNA, genomic orsynthetic DNA libraries for related sequences. Regions of the DNA and/oramino acid sequence of Fhm polypeptide that exhibit significant identityto known sequences are readily determined using sequence alignmentalgorithms as described herein, and those regions may be used to designprobes for screening.

[0099] The term “highly stringent conditions” refers to those conditionsthat are designed to permit hybridization of DNA strands whose sequencesare highly complementary, and to exclude hybridization of significantlymismatched DNAs. Hybridization stringency is principally determined bytemperature, ionic strength, and the concentration of denaturing agentssuch as formamide. Examples of “highly stringent conditions” forhybridization and washing are 0.015 M sodium chloride, 0.0015. M sodiumcitrate at 65-68° C. or 0.015 M sodium chloride, 0.0015 M sodiumcitrate, and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, (Cold Spring Harbor, N.Y. 1989); and Anderson et al.,Nucleic Acid Hybridization: a Practical approach, Ch. 4, IRL PressLimited (Oxford, England). Limited, Oxford, England. More stringentconditions (such as higher temperature, lower ionic strength, higherformamide, or other denaturing agent) may also be used, used; however,the rate of hybridization will be affected. Other agents may be includedin the hybridization and washing buffers for the purpose of reducingnon-specific and/or background hybridization. Examples are 0.1% bovineserum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate,0.1% sodium dodecylsulfate (NaDodSO4 or SDS), ficoll, Denhardt'ssolution, sonicated salmon sperm DNA (or another non-complementary DNA),and dextran sulfate, although other suitable agents can also be used.The concentration and types of these additives can be changed withoutsubstantially affecting the stringency of the hybridization conditions.Hybridization experiments are usually carried out at pH 6.8-7.4,6.8-7.4; however, at typical ionic strength conditions, the rate ofhybridization is nearly independent of pH. See Anderson et al., NucleicAcid Hybridization: a Practical Approach, Ch. 4, IRL Press Limited(Oxford, England).

[0100] Factors affecting the stability of a DNA duplex include basecomposition, length, and degree of base pair mismatch. Hybridizationconditions can be adjusted by one skilled in the art in order toaccommodate these variables and allow -DNAs of different sequencerelatedness to form hybrids. The melting temperature of a perfectlymatched DNA duplex can be estimated by the following equation:

Tm(° C.)=81.5+16.6(log [Na+])+0.41(% G+C)−600/N−0.72(% formamide)

[0101] where N is the length of the duplex formed, [Na+] is the molarconcentration of the sodium ion in the hybridization or washingsolution, % G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, the melting temperature isreduced by approximately 1° C. for each 1% mismatch.

[0102] The term “moderately” stringent conditions” “refers to conditionsunder which a DNA duplex with a greater degree of base pair mismatchingthan could occur under “highly stringent conditions” is able to form.Examples of typical “moderately stringent conditions” are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 50-65° C. or 0.015 M sodiumchloride, 0.0015 M sodium citrate, and 20% formamide at 37-50° C. Bywayof example, a “moderately stringent” condition of 50° C. in 0.015 Msodium ion will allow about a 21% mismatch.

[0103] It will be appreciated by those skilled in the art that there isno absolute distinction between “highly” and “moderately” stringentconditions. For example, at 0.015M sodium ion (no formamide), themelting temperature of perfectly matched long DNA is about 71° C. With awash at 65° C. (at the same ionic strength), this would allow forapproximately a 6% mismatch. To capture more distantly relatedsequences, one skilled in the art can simply lower the temperature orraise the ionic strength.

[0104] A good estimate of the melting temperature in 1M NaCl* foroligonucleotide probes up to about 20 nt is given by:

Tm=2° C. per A-T base pair+4° C. per G-C base pair

[0105] *The sodium ion concentration in 6× salt sodium citrate (SSC) is1 M. See Suggs et al., Developmental Biology Using Purified Genes,p.683, Brown and Fox (eds.) (1981). High stringency washing conditionsfor oligonucleotides are usually at a temperature of 0-5° C. below theTm of the oligonucleotide in 6×SSC, 0.1% SDS.

[0106] In another embodiment, related nucleic acid molecules comprise orconsist of a nucleotide sequence that is about 70 percent (70%)identical to the nucleotide sequence as shown in SEQ ID NO: 3, orcomprise or consist essentially of a nucleotide sequence encoding apolypeptide that is about 70 percent (70%) identical to the polypeptideas set forth in SEQ ID NO: 4. In preferred embodiments, the nucleotidesequences are about 75 percent, or about 80 percent, or about 85percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percentidentical to the nucleotide sequence as shown in SEQ ID NO: 3, or thenucleotide sequences encode a polypeptide that is about 75 percent, orabout 80 percent, or about 85 percent, or about 90 percent, or about 95,96, 97, 98, or 99 percent identical to the polypeptide sequence as setforth in SEQ ID NO: 4.

[0107] Differences in the nucleic acid sequence may result inconservative and/or non-conservative modifications of the amino acidsequence relative to the amino acid sequence of SEQ ID NO: 4.

[0108] Conservative modifications to the amino acid sequence of SEQ IDNO: 4 (and corresponding modifications to the encoding nucleotides) willproduce Fhm polypeptides having functional and chemical characteristicssimilar to those of a naturally occurring Fhm polypeptide. In contrast,substantial modifications in the functional and/or chemicalcharacteristics of Fhm polypeptides may be accomplished by selectingsubstitutions in the amino acid sequence of SEQ ID NO: 4 that differsignificantly in their effect on maintaining (a) the structure of themolecular backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

[0109] For example, a “conservative amino acid substitution”“conservative amino acid substitution” may involve a substitution of anative amino acid residue with a nonnative residue such that there islittle or no effect on the polarity or charge of the amino acid residueat that position. Furthermore, any native residue in the polypeptide mayalso be substituted with alanine, as has been previously described for“alanine scanning mutagenesis.”

[0110] Naturally occurring residues may be divided into groups based oncommon side chain properties:

[0111] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0112] 2) neutral hydrophilic: Cys, Ser, Thr;

[0113] 3) acidic: Asp, Glu;

[0114] 4) basic: Asn, Gln, His, Lys, Arg;

[0115] 5) residues that influence chain orientation: Gly, Pro; and

[0116] 6) aromatic: trp, Tyr, Phe.

[0117] Non-conservative substitutions may involve the exchange of amember of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of the human Fhmpolypeptide that are homologous with non-human Fhm polypeptideorthologs, or into the non-homologous regions of the molecule.

[0118] In making such changes, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of its hydrophobicity and charge characteristics. They are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (-0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

[0119] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131, 1982. It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

[0120] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. The greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

[0121] The following hydrophilicity values have been assigned to theseamino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5);(−2.5) and tryptophan (−3.4). In making changes based uponsimilar hydrophilicity values, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred. One may also identify epitopes from primaryamino acid sequences on the basis of hydrophilicity. These regions arealso referred to as “epitopic core regions.”

[0122] Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the Fhmpolypeptide, or to increase or decrease the affinity of the Fhmpolypeptides described herein.

[0123] Exemplary amino acid substitutions are set forth in Table I.TABLE I Amino Acid Substitutions Original Residues ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn GluAsp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met,Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe LysArg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu PheLeu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr SerSer Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe,Leu Ala, Norleucine

[0124] A skilled artisan will be able to determine suitable variants ofthe polypeptide as set forth in SEQ ID NO: 4 using well knowntechniques. For identifying suitable areas of the molecule that may bechanged without destroying activity, one skilled in the art may targetareas not believed to be important for activity. For example, whensimilar polypeptides with similar activities from the same species orfrom other species are known, one skilled in the art may compare theamino acid sequence of an Fhm polypeptide to such similar polypeptides.With such a comparison, one can identify residues and portions of themolecules that are conserved among similar polypeptides. It will beappreciated that changes in areas of an Fhm polypeptide that are notconserved relative to such similar polypeptides would be less likely toadversely affect the biological activity and/or structure of the Fhmpolypeptide. One skilled in the art would also know that, even inrelatively conserved regions, one may substitute chemically similaramino acids for the naturally occurring residues while retainingactivity (conservative amino acid residue substitutions). Therefore,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

[0125] Additionally, one skilled in the art can reviewstructure-function studies identifying residues in similar polypeptidesthat are important for activity or structure. In view of such acomparison, one can predict the importance of amino acid residues in anFhm polypeptide that correspond to amino acid residues that which areimportant for activity or structure in similar polypeptides. One skilledin the art may opt for chemically similar amino acid substitutions forsuch predicted important amino acid residues of Fhm polypeptides.

[0126] One skilled in the art can also analyze the three-dimensionalstructure and amino acid sequence in relation to that structure insimilar polypeptides. In view of such information, one skilled in theart may predict the alignment of amino acid residues of an Fhmpolypeptide with respect to its three dimensional structure. One skilledin the art may choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each desired amino acid residue. The variantscan then be screened using activity assays know to those skilled in theart. Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change would be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

[0127] A number of scientific publications have been devoted to theprediction of secondary structure. (See Moult J., Curr. Op. in Biotech.,7(4):422-427, 1996. Chou et al., Biochemistry, 13(2):222-245, 1974; Chouet al., Biochemistry, 113(2):211-222, 1974; Chou et al., Adv. Enzymol.Relat. Areas Mol. Biol., 47:45-148, 1978; Chou et al., Ann. Rev.Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384, 1979).Moreover, computer programs are currently available to assist withpredicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural data base (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1):244-247, 1999. It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376, 1997) that thereare a limited number of folds in a given polypeptide or protein and thatonce a critical number of structures have been resolved, structuralprediction will become dramatically in more accurate.

[0128] Additional methods of predicting secondary structure include“threading” (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87, 1997;Sippl et al., Structure, 4(l):15-9, 1996), “profile analysis” (Bowie etal., Science, 253:164-170, 1991); Gribskov et al., Meth. Enzym.,183:146-159, 1990; Gribskov et al., Proc. Nat. Acad. Sci.,84(13):4355-4358 1987), and “evolutionary linkage” (See Home, supra, andBrenner, supra).

[0129] Preferred Fhm polypeptide variants include glycosylation variantswherein the number and/or type of glycosylation sites has been alteredcompared to the amino acid sequence set forth in SEQ ID NO: 4. In oneembodiment, Fhm polypeptide variants comprise a greater or a lessernumber of N-linked glycosylation sites than the amino acid sequence setforth in SEQ ID NO: 4. An N-linked glycosylation site is characterizedby the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residuedesignated as X may be any amino acid residue except proline. Thesubstitution(s) of amino acid residues to create this sequence providesa potential new site for the addition of an N-linked carbohydrate chain.Alternatively, substitutions which eliminate this sequence will removean existing N-linked carbohydrate chain. Also provided is arearrangement of N-linked carbohydrate chains wherein one or moreN-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated.

[0130] Additional preferred Fhm variants include cysteine variants,wherein one or more cysteine residues are deleted from or substitutedfor another amino acid (e.g., serine) as compared to the amino acidsequence set forth in SEQ ID NO: 4. Cysteine variants are useful whenFhm polypeptides must be refolded into a biologically activeconformation such as after the isolation of insoluble inclusion bodies.Cysteine variants generally have fewer cysteine residues than the nativeprotein, and typically have an even number to minimize interactionsresulting from unpaired cysteines.

[0131] In addition, the polypeptide comprising the amino acid sequenceof SEQ ID NO: 4 or an Fhm polypeptide variant may be fused to ahomologous polypeptide to form a homodimer or to a heterologouspolypeptide to form a heterodimer. Heterologous peptides andpolypeptides include, but are not limited to: an epitope to allow forthe detection and/or isolation of an Fhm fusion polypeptide; atransmembrane receptor protein or a portion thereof, such as anextracellular domain, or a transmembrane and intracellular domain; aligand or a portion thereof which binds to a transmembrane receptorprotein; an enzyme or portion thereof which is catalytically active; apolypeptide or peptide which promotes oligomerization, such as a leucinezipper domain; a polypeptide or peptide which increases stability, suchas an immunoglobulin constant region; and a polypeptide which has atherapeutic activity different from the polypeptide comprising the aminoacid sequence as set forth in SEQ ID NO: 4 or an Fhm polypeptidevariant.

[0132] Fusions can be made either at the amino terminus or at thecarboxy terminus of the polypeptide comprising the amino acid sequenceset forth in SEQ ID NO: 4 or an Fhm polypeptide variant. Fusions may bedirect with no linker or adapter molecule, or indirect using a linker oradapter molecule. A linker or adapter molecule may be one or more aminoacid residues, typically up to from about 20 to about 50 amino acidresidues. A linker or adapter molecule may also be designed with acleavage site for a DNA restriction endonuclease or for a protease toallow for the separation of the fused moieties. It will be appreciatedthat once constructed, the fusion polypeptides can be derivatizedaccording to the methods described herein.

[0133] In a further embodiment of the invention, the polypeptidecomprising the amino acid sequence of SEQ ID NO: 4 or an Fhm polypeptidevariant is fused to one or more domains of an Fc region of human IgG.Antibodies comprise two functionally independent parts, a variabledomain known as “Fab”, which binds antigens, and a constant domain knownas “Fc”, which is involved in effector functions such as complementactivation and attack by phagocytic cells. An Fc has a long serumhalf-life, whereas an Fab is short-lived. (Capon et al., Nature,337:525-31, 1989). When constructed together with a therapeutic protein,an Fc domain can provide longer half-life or incorporate such functionsas Fc receptor binding, protein A binding, complement fixation andperhaps even placental transfer. Id. Table II summarizes the use ofcertain Fc fusions known in the art. TABLE II Fc Fusion with TherapeuticProteins Fusion Therapeutic Form of Fc partner implications ReferenceIgG1 N-terminus Hodgkin's disease; U.S. Pat. No. of CD30-L anaplastic5,480,981 lymphoma; T-cell leukemia Murine IL-10 anti-inflammatory;Zheng et al. Fcg2a transplant rejection (1995), J. Immunol., 154:5590-5600 IgG1 TNF septic shock Fisher et al. receptor (1996), N. Engl.J. Med., 334: 1697- 1702; VanZee et al., (1996), J. Immunol., 156:2221-2230 IgG, IgA, TNF inflammation, U.S. Pat. No. IgM, or IgE receptorautoimmune 5,808,029, issued (excluding disorders Sep. 15, 1998 thefirst domain) IgG1 CD4 AIDS Capon et al. receptor (1989), Nature 337:525-531 IgG1, N-terminus anti-cancer, antiviral Harvill et al. IgG3 ofIL-2 (1995), Immunotech., 1: 95-105 IgG1 C-terminus osteoarthritis; WO97/23614, of OPG bone density published Jul. 3, 1997 IgG1 N-terminusanti-obesity PCT/US of leptin 97/23183, filed Dec. 11, 1997 Human IgCTLA-4 autoimmune Linsley (1991), J. Cg1 disorders Exp. Med.,174:561-569

[0134] In one example, all or a portion of the human IgG hinge, CH2 andCH3 regions may be fused at either the N-terminus or C-terminus of theFhm polypeptides using methods known to the skilled artisan. Theresulting Fhm fusion polypeptide may be purified by use of a Protein Aaffinity column. Peptides and proteins fused to an Fc region have beenfound to exhibit a substantially greater half-life in vivo than theunfused counterpart. Also, a fusion to an Fc region allows fordimerization/multimerization of the fusion polypeptide. The Fc regionmay be a naturally occurring Fc region, or may be altered to improvecertain qualities, such as therapeutic qualities, circulation time,reduce aggregation, etc.

[0135] Identity and similarity of related nucleic acid molecules andpolypeptides can be readily calculated by known methods. Such methodsinclude, but are not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, NewYork,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W.,ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data,Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, NewJersey, 1994; Sequence Analysis in Molecular Biolog, von Heinje, G.,Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M. Stockton Press, New York, 1991; and Carillo etal., SIAM J. Applied Math. 48:1073, 1988.

[0136] Preferred methods to determine identity and/or similarity aredesigned to give the largest match between the sequences tested. Methodsto determine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,Nucl. Acid. Res., 12:387, 1984; Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,J. Mol. Biol., 215:403-410, 1990). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda,Md. 20894; Altschul et al., supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

[0137] Certain alignment schemes for aligning two amino acid sequencesmay result in the matching of only a short region of the two sequences,and this small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method (GAP program) will result in an alignment that spans atleast 50 contiguous amino acids of the target polypeptide.

[0138] For example, using the computer algorithm GAP (Genetics ComputerGroup, University of Wisconsin, Madison, Wis.), two polypeptides forwhich the percent sequence identity is to be determined are aligned foroptimal matching of their respective amino acids (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3× the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually {fraction (1/10)} times the gap opening penalty), as well as acomparison matrix such as PAM 250 or BLOSUM 62 are used in conjunctionwith the algorithm. A standard comparison matrix (see Dayhoff et al.,Atlas of Protein Sequence and Structure, vol. 5, supp.35(3), 1978 forthe PAM 250 comparison matrix; Henikoff et al., Proc. Natl. Acad. SciUSA, 89:10915-10919, 1992 for the BLOSUM 62 comparison matrix) is alsoused by the algorithm.

[0139] Preferred parameters for polypeptide sequence comparison includethe following:

[0140] Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970),

[0141] Comparison matrix: BLOSUM 62 from Henikoff and Henikoff, Proc.Natl. Acad. Sci. USA 89:10915-10919 (1992).

[0142] Gap Penalty: 12

[0143] Gap Length Penalty: 4

[0144] Threshold of Similarity: 0

[0145] The GAP program is useful with the above parameters. Theaforementioned parameters are the default parameters for polypeptidecomparisons (along with no penalty for end gaps) using the GAPalgorithm.

[0146] Preferred parameters for nucleic acid molecule sequencecomparison include the following:

[0147] Algorithm: Needleman and Wunsch, J. Mol Biol. 48:443-4, 1970

[0148] Comparison matrix: matches=+10, mismatch=0

[0149] Gap Penalty: 50

[0150] Gap Length Penalty: 3

[0151] The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

[0152] Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused by those of skill in the art, including those set forth in theProgram Manual, Wisconsin Package, Version 9, September, 1997. Theparticular choices to be made will be apparent to those of-skill in theart and will depend on the specific comparison to be made, such asDNA-to-DNA, protein-to-protein, protein-to-DNA; and additionally,whether the comparison is between pairs of sequences (in which case GAPor BestFit are generally preferred) or between one sequence and a largedatabase of sequences (in which case FASTA or BLASTA are preferred).

[0153] Synthesis

[0154] It will be appreciated by those skilled in the art the nucleicacid and polypeptide molecules described herein may be produced byrecombinant and other means.

[0155] Nucleic Acid Molecules

[0156] The nucleic acid molecules encode a polypeptide comprising theamino acid sequence of an Fhm polypeptide and can readily be obtained ina variety of ways including, without limitation, chemical synthesis,cDNA or genomic library screening, expression library screening and/orPCR amplification of cDNA

[0157] Recombinant DNA methods used herein are generally, those setforth in Sambrook et al. (Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and/orAusubel et al., eds., (Current Protocols in Molecular Biology, GreenPublishers Inc. and Wiley and Sons, NY (1994)).

[0158] The present invention provides for nucleic acid molecules asdescribed herein and methods for obtaining such molecules. Where a geneencoding Fhm polypeptide has been identified from one species, all or aportion of that gene may be used as a probe to identify orthologs orrelated genes from the same species. The probes or primers may be usedto screen cDNA libraries from various tissue sources believed to expressthe Fhm polypeptide

[0159] In addition, part or all of a nucleic acid molecule having thesequence as set forth in SEQ ID NO: 3 may be used to screen a genomiclibrary to identify and isolate a gene encoding Fhm. Typically,conditions of moderate or high stringency will be employed for screeningto minimize the number of false positives obtained from the screen.

[0160] Nucleic acid molecules encoding the amino acid sequence of Fhmpolypeptides may also be identified by expression cloning, which employsthe detection of positive clones based upon a property of the expressedprotein. Typically, nucleic acid libraries are screened by the bindingof an antibody or other binding partner (e.g., receptor or ligand) tocloned proteins which are expressed and displayed on a host cellsurface. The antibody or binding partner is modified with a detectablelabel to identify those cells expressing the desired clone.

[0161] Recombinant expression techniques conducted in accordance withthe descriptions set forth below may be followed to produce thesepolynucleotides and to express the encoded polypeptides. For example, byinserting a nucleic acid sequence which encodes the amino acid sequenceof an Fhm polypeptide into an appropriate vector, one skilled in the artcan readily produce large quantities of the desired nucleotide sequence.The sequences can then be used to generate detection probes oramplification primers. Alternatively, a polynucleotide encoding theamino acid sequence of an Fhm polypeptide can be inserted into anexpression vector. By introducing the expression vector into anappropriate host, the encoded Fhm polypeptide may be produced in largeamounts.

[0162] Another method for obtaining a suitable nucleic acid sequence isthe polymerase chain reaction (PCR). In this method, cDNA is preparedfrom poly(A)+RNA or total RNA using the enzyme reverse transcriptase.Two primers, typically complementary to two separate regions of cDNA(oligonucleotides) encoding the amino acid sequence of an Fhmpolypeptide, are then added to the cDNA along with a polymerase such asTaq polymerase, and the polymerase amplifies the cDNA region between thetwo primers.

[0163] Another means of preparing a nucleic acid molecule encoding theamino acid sequecne of Fhm polypeptide is by chemical synthesis usingmethods well known to the skilled artisan such as those described byEngels et al., Angew. Chem. Intl. Ed., 28:716-734, 1989. These methodsinclude, inter alia, the phosphotriester, phosphoramidite, andH-phosphonate methods for nucleic acid synthesis. A preferred method forsuch chemical synthesis is polymer-supported synthesis using standardphosphoramidite chemistry. Typically, the DNA encoding the amino acidsequence of a Fhm polypeptide will be several hundred nucleotides inlength. Nucleic acids larger than about 100 nucleotides can besynthesized as several fragments using these methods. The fragments canthen be ligated together to form the full-length nucleotide sequence ofa Fhm polypeptide. Usually, the DNA fragment encoding the amino terminusof the polypeptide will have an ATG, which encodes a methionine residue.This methionine may or may not be present on the mature form of the Fhmpolypeptide, depending on whether the polypeptide produce din the hostcell is designed to be secreted from the cell. Other methods known tothe skilled artisan may be used as well.

[0164] In certain embodiments, nucleic acid variants contain codonswhich have been altered for the optimal expression of a Fhm polypeptidein a given host cell. Particular codon alterations will depend upon theFhm polypeptide(s) and host cell(s) selected for expression. Such “codonoptimization” can be carried out by a variety of methods, for example,by selecting codons which are preferred for use in highly expressedgenes in a given host cell. Computer algorithms which incorporate codonfrequency tables such as “Ecohigh. cod” for codon preference of highlyexpressed bacterial genes may be used and are provided by the Universityof Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.Other useful codon frequency tables include “Celegans_high.cod”,“Celegans_low.cod”, “Drosophila_high.cod”, “Human_high.cod”,“Maize_high.cod”, and “Yeast_high.cod”.

[0165] In other embodiments, nucleic acid molecules encode Fhm variantswith conservative amino acid substitutions as defined above, Fhmvariants comprising an addition and/or a deletion of one or moreN-linked or O-linked glycosylation sites, or Fhm polypeptide fragmentsas described above. In addition, nucleic acid molecules may encode anycombination of Fhm variants, fragments, and fusion polypeptidesdescribed herein provided that DNA's modified in this way code forpolypeptides capable of finding one or more members of TNF supergenefamily of ligands and receptors.

[0166] Vectors and Host Cells

[0167] A nucleic acid molecule encoding the amino acid sequence of a Fhmpolypeptide may be inserted into an appropriate expression vector usingstandard ligation techniques. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). A nucleic acid moleculeencoding the amino acid sequence of a Fhm polypeptide may beamplified/expressed in prokaryotic, yeast, insect (baculovirus systems),and/or eukaryotic host cells. Selection of the host cell will depend inpart on whether the Fhm polypeptide is to be post-translationallymodified (e.g., glycosylated and/or phosphorylated). If so, yeast,insect, or mammalian host cells are preferable. For a review ofexpression vectors see Meth. Enz. vol. 185 D. V. Goeddel ed., AcademicPress, Sna Diego Calif., 1990.

[0168] Typically, expression vectors used in any of the host cells willcontain sequences for plasmid maintenance and for cloning and expressionof exogenous nucleotide sequences. Such sequences, collectively referredto as “flanking sequences” (in certain embodiments will typicallyinclude one or more of the following nucleotide sequences: a promoter,one or more enhancer sequences, an origin of replication, atranscriptional termination sequence, a complete intron sequencecontaining a donor and acceptor splice site, a sequence encoding aleader sequence for secretion, a ribosome binding site, apolyadenylation sequence, a polylinker region for inserting the nucleicacid encoding the polypeptide to be expressed, and a selectable markerelement. Each of these sequences is discussed below.

[0169] Optionally, the vector may contain a “tag” sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the Fhmpolypeptide coding sequence; the oligonucleotide molecule encodespolyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus) or myc for which commercially availableantibodies exist. Optionally, the Fhm gene can also be fused in frame atthe N-terminal for example to an IgG Fc region. This tag is typicallyfused to the polypeptide upon expression of the polypeptide, and canserve as a means for affinity purification of the Fhm polypeptide fromthe host cell although it may also prolong the circulatory half life ofa Fhm polypeptide. Affinity purification can be accomplished, forexample, by column chromatography using antibodies against the tag as anaffinity matrix. Optionally, the tag can subsequently be removed fromthe purified Fhm polypeptide by various means such as using certainpeptidases for cleavage.

[0170] Flanking sequences may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof flanking sequences from more than one source), or synthetic, ortehflanking sequence may be native sequences which normally function toregulate Fhm expression. As such, the source of flanking sequences maybe any prokaryotic or eukaryotic organism, any vertebrate orinvertebrate organism, or any plant, provided that a flanking sequenceis functional in, and can be activated by, the host cell machinery.

[0171] The flanking sequences useful in the vectors of this inventionmay be obtained by any of several methods well known in the art.Typically, flanking sequences useful herein other than the sequencesflanking the Fhm gene will have been previously identified by mappingand/or by restriction endonuclease digestion and can thus be isolatedfrom the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

[0172] Where all or only a portion of the flanking sequence is known, itmay be obtained using PCR and/or by screening a genomic library withsuitable oligonucleotide and/or flanking sequence fragments from thesame or another species.

[0173] Where the flanking sequence is not known, a fragment of DNAcontaining a flanking sequence may be isolated from a larger piece ofDNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion to produce the proper DNA fragment followed by isolation usingagarose gel purification, Qiagen® column chromatography (Chatsworth,Calif.), or other method known to the skilled artisan. The selection ofsuitable enzymes to accomplish this purpose will be readily apparent toone of ordinary skill in the art.

[0174] An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. Amplification of the vectorto a certain copy number can, in some cases, be important for theoptimal expression of the Fhm polypeptide. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (Product No.303-3s, New England Biolabs, Beverly, Mass.) is suitable for mostGram-negative bacteria and various origins (e.g., SV40, polyoma,adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses such asHPV or BPV) are useful for cloning vectors in mammalian cells.Generally, the origin of replication component is not needed formammalian expression vectors (for example, the SV40 origin is often usedonly because it contains the early promoter).

[0175] A transcription termination sequence is typically located 3′ ofthe end of a polypeptide coding regions and serves to terminatetranscription. Usually, a transcription termination sequence inprokaryotic cells is a G-C rich fragment followed by a poly T sequence.While the sequence is easily cloned from a library or even purchasedcommercially as part of a vector, it can also be readily synthesizedusing methods for nucleic acid synthesis such as those described herein.

[0176] A selectable marker gene element encodes a protein necessary forthe survival and growth of a host cell grown in a selective culturemedium. Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

[0177] Other selection genes may be used to amplify the gene which willbe expressed. Amplification is the process wherein genes which are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure whichonly the transformants are uniquely adapted to survive by virtue of theselection gene present in the vector. Selection pressure is imposed byculturing the transformed cells under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to the amplification of both the selection gene and theDNA that encodes Fhm. As a result, increased quantities of Fhm aresynthesized from the amplified DNA.

[0178] A ribosome binding site is usually necessary for translationinitiation of mRNA and is characterized by a Shine-Dalgamo sequence(prokaryotes) or a Kozak sequence (eukaryotes). The element is typicallylocated 3′ to the promoter and 5′ to the coding sequence of the Fhmpolypeptide to be expressed. The Shine-Dalgamo sequence is varied but istypically a polypurine (i.e., having a high A-G content). ManyShine-Dalgamo sequences have been identified, each of which can bereadily synthesized using methods set forth herein and used in aprokaryotic vector.

[0179] A leader, or signal, sequence may be used to direct the secretionof Fhm polypeptide out of the host cell where it is synthesized.Typically, a nucleotide sequence encoding the signal sequence ispositioned in the coding region of the Fhm nucleic acid molecule, ordirectly at the 5′ end of the Fhm polypeptide coding region. Many signalsequences have been identified, and any of those that are functional inthe selected host cell may be used in conjunction with the Fhm gene orcDNA. Therefore, a signal sequence may be homologous (naturallyoccurring) or heterologous to the Fhm gene or cDNA, and may behomologous or heterologous to the Fhm gene or cDNA. Additionally, asignal sequence may be chemically synthesized using methods describedherein. In most cases, the secretion of an Fhm polypeptide from the hostcell via the presence of a signal peptide will result in the removal ofthe signal peptide from the Fhm polypeptide.

[0180] The signal sequence may be a component of the vector, or it maybe a part of Fhm nucleic acid molecule that is inserted into the vector.The native Fhm DNA encodes a signal sequence at the amino terminus ofthe protein that is cleaved during post-translational processing of themolecule to form the mature Fhm protein product. Included within thescope of this invention are Fhm nucleotides with the native signalsequence as well as Fhm nucleotides wherein the native signal sequenceis deleted and replaced with a heterologous signal sequence. Theheterologous signal sequence selected should be one that isrecognized-and processed, i.e., cleaved by a signal peptidase, by thehost cell. For prokaryotic host cells that do not recognize and processthe native Fhm signal sequence, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, or heat-stable enterotoxin IIleaders. For yeast secretion, the native Fhm signal sequence may besubstituted by the yeast invertase, alpha factor, or acid phosphatasesignal sequences. For mammalian cell expression the native signalsequence of the Fhm polypeptideis satisfactory, although other mammaliansignal sequences may be suitable.

[0181] In some cases, such as where glycosylation is desired in aeukaryotic host cell expression system, one may manipulate the variouspresequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addpresequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein), one or more additional amino acid residuesincident to expression, which may not have been totally removed. Forexample, the final protein product may have one or two amino acids foundin the peptidase cleavage site, attached to the N-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired Fhm polypeptide, if the enzymecuts at such area within the mature polypeptide.

[0182] In many cases, transcription of a nucleic acid molecule isincreased by the presence of one or more introns in the vector; this isparticularly true where a polypeptide is produced in eukaryotic hostcells, especially mammalian host cells. The introns used may benaturally occurring within the Fhm gene, especially where the gene usedis a full length genomic sequence or a fragment thereof Where the intronis not naturally occurring within the gene (as for most cDNAs), theintron(s) may be obtained from another source. The position of theintron with respect to flanking sequences and the Fhm gene is generallyimportant, as the intron must be transcribed to be effective. Thus, whenan Fhm cDNA molecule is being transcribed, the preferred position forthe intron is 3′ to the transcription start site, and 5′ to the polyAtranscription termination sequence. Preferably, the intron or intronswill be located on one side or the other (i.e., 5′ or 3′) of the cDNAsuch that it does not interrupt the this coding sequence. Any intronfrom any source, including viral, prokaryotic and eukaryotic (plant oranimal) organisms, may be used to practice this invention, provided thatit is compatible with the host cell(s) into which it is inserted. Alsoincluded herein are synthetic introns. Optionally, more than one intronmay be used in the vector.

[0183] The expression and cloning vectors of the present invention willeach typically contain a promoter that is recognized by the hostorganism and operably linked to the molecule encoding the Fhmpolypeptide.

[0184] Promoters are untranscribed sequences located upstream (5′) tothe start codon of a structural gene (generally within about 100 to 1000bp) that control the transcription and translation of a particularmolecule, such as that encoding Fhm. Promoters are conventionallygrouped into one of two classes, inducible promoters and constitutivepromoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the othere hand,initiate continuous gene production; that is, there is little or nocontrol over gene expression. A large number of promoters, recognized bya variety of potential host cells, are well known. A suitable promoteris operably linked to the DNA encoding Fhm by removing the promoter fromthe source DNA by restriction enzyme digestion and inserting the desiredpromoter sequence into the vector. The native Fhm promoter sequence maybe used to direct amplification and/or expression of Fhm encodingnucleic acid molecule. A heterologous promoter is preferred, however, ifit permits greater transcription and higher yields of the expressedprotein as compared to the native promoter, and if it is compatible withthe host cell system that has been selected for use.

[0185] Promoters suitable for use with prokaryotic hosts include, butare not limited to the beta-lactamase and lactose promoter systems;alkaline phosphatase, a tryptophan (trp) promoter system; and hybridpromoters such as the tac promoter. Other known bacterial promoteres andalso suitable. Their sequences have been published, thereby enabling oneskilled in the art to ligate them to the desired DNA sequence(s), usinglinkers or adapters as needed to supply any useful restriction sites.

[0186] Suitable promoters for use with yeast hosts are also well knownin the art. Yeast enhancers are advantageously used with yeastpromoters. Suitable promoters for use with mammalian host cells are wellknown and include, but are not limited to, those obtained from thegenomes of viruses such as polyoma virus, fowl pox virus, adenovirus(such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retrovirus, hepatitis-B virus, herpes virus and mostpreferably Simian Virus 40 (SV40). Other suitable mammalian promotersinclude heterologous mammalian promoters, e.g., heat-shock promoters andthe actin promoter.

[0187] Additional promoters which may be of interest in controlling Fhmtranscription include, but are not limited to, the SV40 early promoterregion (Bemoist and Chambon, Nature, 290:304-310, 1981); the CMVpromoter; the promoter contained in the 3′ long terminal repeat (LTR) ofRous sarcoma virus (RSV) (Yamamoto, et al., Cell, 22:787-797, 1980); theherpes thymidine kinase (TK) promoter (Wagner et al, Proc. Natl. Acad.Sci. U.S.A., 78:144-1445, 1981); the regulatory sequences of themetallothionine gene (Brinster et al., Nature, 296:39-42, 1982);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. U.S.A., 75:3727-3731,1978); or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci.U.S.A., 80:21-25, 1983). Also of use are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion which is active in pancreatic acinar cells (Swift et al., Cell,38:639-646, 1984; Ornitz et al., Cold Spring Harbor Symp. Quant. Biol.50:399-409, 1986; MacDonald, Hepatology, 7:425-515, 1987); the insulingene control region which is active in pancreatic beta cells (Hanahan,Nature, 315:115-122, 1985); the immunoglobulin gene control region whichis active in lymphoid cells (Grosschedl et al., Cell, 38:647-658, 1984;Adames et al., Nature, 318:533-538, 1985; Alexander et al., Mol. Cell.Biol., 7:1436-1444, 1987); the mouse mammary tumor virus control regionwhich is active in testicular, breast, lymphoid and mast cells (Leder etal., Cell, 45:485-495, 1986), the albumin gene control region which isactive in liver (Pinkert et al., Genes and Devel., 1:268-276, 1987); thealphafetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol., 5:1639-1648, 1985; Hammer et al., Science,235:53-58, 1987); the alpha 1-antitrypsin gene control region which isactive in the liver (Kelsey et al., Genes and Devel., 1:161-171, 1987);the beta-globin gene control region which is active in myeloid cells(Mogram et al., Nature, 315:338-340, 1985; Kollias et al., Cell,46:89-94, 1986); the myelin basic protein gene control region which isactive in oligodendrocyte cells in the brain (Readhead et al., Cell,48:703-712, 1987); the myosin light chain-2 gene control region which isactive in skeletal muscle (Sani, Nature, 314:283-286, 1985); and thegonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., Science, 234:1372-1378, 1986).

[0188] An enhancer sequence may be inserted into the vector to increasethe transcription of a DNA encoding a Fhm polypeptide of the presentinvention by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease its transcription. Enhancers are relatively orientation andposition independent. They have been found 5′ and 3′ to thetranscription unit. Several enhancer sequences available from mammaliangenes are known (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin). Typically, however, an enhancer from a virus will be used. TheSV40 enhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer, and adenovirus enhancers are exemplary enhancing elements forthe activation or upregulation of eukaryotic promoters. While anenhancer may be spliced into the vector at a position 5′ or 3′ to Fhmnucleic acid molecules, it is typically located at a site 5′ from thepromoter.

[0189] Expression vectors of the invention may be constructed from astarting vector such as a commercially available vector. Such vectorsmay or may not contain all of the desired flanking sequences. Where oneor more of the desired flanking sequences set forth above are notalready present in the vector, they may be individually obtained andligated into the vector. Methods used for-obtaining each of the flankingsequences are well known to one skilled in the art.

[0190] Preferred vectors for practicing this invention are those whichare compatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII. pCR3, and pcDNA3.1 (InvitrogenCompany, Carlsbad, Calif.), pBSII (Stratagene Company, La Jolla,Calif.), pETI5 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBacII; Invitrogen), pDSR-alpha (PCT Publ. No. WO 90/14364) andpFastBacDual (Gibco-Brl Grand Island, N.Y.).

[0191] Additional suitable vectors include, but are not limited to,cosmids, plasmids or modified viruses, but it will be appreciated thatthe vector system must be compatible with the selected host cell. Suchvectors include, but are not limited to plasmids such as Bluescript®plasmid derivatives (a high copy number ColE1-based phagemid, StratageneCloning Systems Inc., La Jolla Calif.), PCR cloning plasmids designedfor cloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit,PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, Calif.), andmammalian, yeast or virus vectors such as a baculovirus expressionsystem (pBacPAK plasmid derivatives, Clontech, Palo Alto, Calif.).

[0192] After the vector has been constructed and a nucleic acid moleculeencoding an Fhm polypeptide has been inserted into the proper site ofthe vector, the completed vector may be inserted into a suitable hostcell for amplification and/or polypeptide expression. The transformationof an expression vector for an Fhm polypeptide into a selected host cellmay be accomplished by well-known methods such as transfection,infection, calcium chloride, electroporation, microinjection,lipofection or the DEAE-dextran method or other known techniques. Themethod selected will in part be a function of the type of host cell tobe used. These methods and other suitable methods are well known to theskilled artisan, and are set forth, for example, in Sambrook et al.,supra.

[0193] Host cells may be prokaryotic host cells (such as E. coli) oreukaryotic host cells (yeast, insect, or vertebrate cells). The hostcell, when cultured under appropriate conditions, synthesizes an Fhmpolypeptide which can subsequently be collected from the culture medium(if the host cell secretes it into the medium) or directly from the hostcell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that desirable or necessaryfor activity, (such as glycosylation or phosphorylation), and ease offolding into a biologically active molecule.

[0194] Yeast and mammalian cells are preferred hosts of the presentinvention. The use of such hosts provides substantial advantages in thatthey can also carry out post-translational peptide modificationsincluding glycosylation. A number of recombinant DNA strategies existwhich utilize strong promoter sequences and high copy number of plasmidswhich can be utilized for production of the desired proteins in thesehosts.

[0195] Yeast recognize leader sequences on cloned mammalian geneproducts and secrete peptides bearing leader sequences (i.e.,pre-peptides). Mammalian cells provide post-translational modificationsto protein molecules including correct folding or glycosylation atcorrect sites.

[0196] Mammalian cells which may be useful as hosts include cells offibroblast origin such as VERO or CHO-K1, and their derivatives. For amammalian host, several possible vector systems are available for theexpression of the desired Fhm protein. A wide variety of transcriptionaland translational regulatory sequences may be employed, depending uponthe nature of the host. The transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, my'sin, etc., may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the genes can be modulated. Usefulsignals are regulatory signals which are temperature-sensitive so thatby varying the temperature, expression can be repressed or initiated, orare subject to chemical regulation, e.g., metabolite.

[0197] As is widely known, translation of eukaryotic mRNA is initiatedat the codon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the desired receptor molecule does notcontain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation of a fusion protein (if the AUG codon is in the samereading- frame as the desired receptor molecule encoding DNA sequence)or a frame-shift mutation (if the AUG codon is not in the same readingframe as the desired Fhm protein encoding sequence).

[0198] The expression of the Fhm proteins can also be accomplished inprocarvotic cells. Preferred prokaryotic hosts include bacteria such asE. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.The most preferred prokaryotic host is E. coli. Bacterial hosts ofparticular interest include E. coli K12 strain 294 (ATCC 31446), E. coliX1776 (ATCC 31537), E. coli W3110 (F⁻, lambda⁻, prototrophic (ATCC27325)), and other enterobacteria (such as Salmonella typhimurium orSerratia marcescens), and various Pseudomonas species. The prokaryotichost must be compatible with the replicon and control sequences in theexpression plasmid.

[0199] To express the desired Fhm protein in a prokaryotic cell (suchas, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.),it is necessary to operably link the desired receptor molecule encodingsequence to a functional prokaryotic promoter. Such promoters may beeither constitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, and the bla promoter of the β-lactamasegene of pBR322, etc. Examples of inducible prokaryotic promoters includethe major right and left promoters, of bacteriophage λ (P_(L) andP_(R)), the trp, recA, lacZ, lacd, gal, and tac promoters of E. coli,the α-amylase (Ulmanen et al., J. Bacteriol. 162:176-182, 1985), theσ-28-specific promoters of B. subtilis (Gilma et al., Gene 32:11-20,1984), the promoters of the bacteriophages of Bacillus (Gryczan, T. J.,In: The Molecular Biology of the Bacilli, Academic Press, Inc., NewYork, 1982), and Streptomyces promoters (Ward et al., Mol. Gen. Genet.203:468-478 1986). Prokaryotic promoters are reviewed by Glick, (J. Ind.Microbiol. 1:277-282, 1987); Cenatiempo, Biochimie 68:505-516, 1986);and Gottesman, Ann. Rev. Genet. 18:415-442 1984).

[0200] Proper expression in a prokaryotic cell also requires thepresence of a ribosome binding site upstream from the gene-encodingsequence. Such ribosome binding sites are disclosed, for example, byGold et al. (Ann. Rev. Microbiol. 35:365-404, 1981).

[0201] The desired Fhm protein encoding sequence and an operably linkedpromoter may be introduced into a recipient prokaryotic or eukaryoticcell either as a non-replicating DNA (or RNA) molecule, which may eitherbe linear or, more preferably, a closed covalent circular molecule.Since such molecules are incapable of autonomous replication, theexpression of the desired receptor molecule may occur through thetransient expression of the introduced sequence. Alternatively,permanent expression may occur through the integration of the introducedsequence into the host chromosome.

[0202] In one embodiment, a vector is employed which is capable ofintegrating the desired gene sequences into the host cell chromosome.Cells which have stably integrated the introduced DNA into theirchromosomes can be selected by also introducing one or more markerswhich allow for selection of host cells which contain the expressionvector. The marker may complement an auxotrophy in the host (such asleu2l, or ura3, which are common yeast auxotrophic markers), biocideresistance, e.g., antibiotics, or heavy metals, such as copper, or thelike. The selectable marker gene can either be directly linked to theDNA gene sequences to be expressed, or introduced into the same cell byco-transfection.

[0203] In a preferred embodiment, the introduced sequence will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors maybe employed for this purpose. Factors of importance in selecting aparticular plasmid or viral vector include, for e.g. the ease with whichrecipient cells that contain the vector may be recognized and selectedfrom those recipient cells which do not contain the vector; the numberof copies of the vector which are desired in a particular host; andwhether it is desirable to be able to “shuttle” the vector between hostcells of different species.

[0204] Any of a series of yeast gene expression systems can also beutilized. Examples of such expression vectors include the yeast 2-microncircle, the expression plasmids YEP13, YVP and YRP, etc., or theirderivatives. Such plasmids are well known in the art (Botstein, et al.,Miami Wntr. Symp. 19:265-274 (1982); Broach, In: The Molecular Biologyof the Yeast Saccharomyces: Life Cycle and Inheritance, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach,Cell 28:203-204 1982).

[0205] For a mammalian host, several possible vector systems areavailable for expression. One class of vectors utilize DNA elementswhich provide autonomously replicating extra-chromosomal plasmids,derived from animal viruses such as bovine papilloma virus, polyomavirus, adenovirus, or SV40 virus. A second class of vectors relies uponthe integration of the desired gene sequences into the host chromosome.Cells which have stably integrated the introduced DNA into theirchromosomes may be selected by also introducing one or more markerswhich allow selection of host cells which contain the expression vector.The marker may provide for prototropy to an auxotrophic host, biocideresistance, e.g., antibiotics, or heavy metals, such as copper or thelike. The selectable marker gene can either be directly linked to theDNA sequences to be expressed, or introduced into the same cell byco-transformation. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include splice signals, as well astranscription promoters, enhancers, and termination signals. The cDNAexpression vectors incorporating such elements include those describedby Okayama, Mol. Cell. Biol. 3:280 1983, and others. Preferredeukaryotic vectors include PWLNEO, PSV2CAT, POG44, PXTI, pSG, pSVK3,pBPV, pMSG, pSVL (Pharmacia).

[0206] Preferred prokaryotic vectors include plasmids such as thosecapable of replication in E. coli such as, for example, pBR322, ColE1,pSC101, pACYC 184, πVX, pQE70, pQE60, pQE9, pBG, pD10, Phage script,psix174, pbmescript SK, pbsks, pNH8A, pNHIBa, pNH18A, pNH46A (SL raregone), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Such plasmids are,for example, disclosed by Maniatis, T., et al. (In: Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1982)). Bacillus plasmids include pC194, pC221, pT127, etc. Suchplasmids are disclosed by Gryczan, T. (In: The Molecular Biology of theBacilli, Academic Press, New York (1982), pp. 307-329). SuitableStreptomyces plasmids include pISJ101 (Kendall, et al., J. Bacteriol.169:4177-4183 1987), and Streptomyces bacteriophages such as φC31(Chater, et al., In: Sixth International Symposium on ActinomycetalesBiology, Akademiai Kaido, Budapest, Hungary, 1986, pp 45-541).Pseudomonas plasmids are reviewed by John, et al. (Rev. Infect. Dis.8:693-704, 1986, and Izaki, K. (Jpn. J. Bacteriol. 33:729-742 1978).However, any other plasmid or vector may be used as long as they arereplicable and viable in the host cell.

[0207] Once the vector or DNA sequence containing the constructs hasbeen prepared for expression, the DNA constructs may be introduced intoan appropriate host. Various techniques may be employed, such as aprotoplast fusion, calcium phosphate precipitation, electroporation orother conventional techniques. After the fusion, the cells are grown inmedia and screened for appropriate activities. Expression of thesequence results in the production of the Fhm protein.

[0208] Suitable host cells or cell lines may be mammalian cells, such asChinese hamster ovary cells (CHO; ATCC No. CCL61), CHO DHFR cells(Urlaub et al. Proc. Natl. Acad. Sci. U.S.A, 97: 4216-4220,1980) humanembryonic kidney (HEK), 293 or 293T cells (ATCC No. CRL 1573), or 3T3cells (ATCC No. CRL920). The selection of suitable mammalian host cellsand methods for transformation, culture, amplification, screening,product production and purification are known in the art. Other suitablemammalian cell lines, are the monkey COS-1 (ATCC No. CRL 1650) and COS-7(ATCC No. CRL 1651) cell lines, and the CV-1 (ATCC No. CCL70) cell line.Further exemplary mammalian host cells include primate cell lines androdent cell lines, including transformed cell lines. Normal diploidcells, cell strains derived from in vitro culture of primary tissue, aswell as primary explants, are also suitable. Candidate cells may begenotypically deficient in the selection gene, or may contain a dominantacting selection gene. Other suitable mammalian cell lines include, butare not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaKhamster cell lines, which are available from the ATCC. Each of thesecell lines is known by and available to those skilled in the art ofprotein expression.

[0209] Similarly useful as host cells suitable for the present inventionare bacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5α (ATCC No. 33694), DH10, and MC1061 (ATCC No. 53330)) arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomycesspp., and the like may also be employed in this method.

[0210] Many strains of yeast cells known to those skilled in the art arealso available as host cells for expression of the polypeptides of thepresent invention. Preferred yeast strains include, for example,Saccharomyces cerevisiae and Pichia pastoris.

[0211] Additionally, where desired, insect cell systems may be utilizedin the methods of the present invention. Such systems are described forexample in Kitts et al. (Biotechniques, 14:810-817, 1993), Lucklow(Curr. Opin. Biotechnol., 4:564-572, 1993) and Lucklow et al. (J.Virol., 67:4566-4579, 1993). Preferred insect cells are Sf-9 and Hi5(Invitrogen, Carlsbad, Calif.).

[0212] One may also use transgenic animals to express glycosylated Fhmpolypeptides. For example, one may use a transgenic milk-producinganimal (e.g. a cow or goat) and obtain the present glycosylatedpolypeptide in the animal milk. One may also use plants to produce Fhmpolypeptides; however, in general, the glycosylation occurring in plantsis different from that produced in mammalian cells, and may result in aglycosylated product which is not suitable for human therapeutic use.

[0213] Polypeptide Production

[0214] Host cells comprising an Fhm polypeptide expression vector (i.e.,transformed or transfected) may be cultured using standard media wellknown to the skilled artisan. The media will usually contain allnutrients necessary for the growth and survival of the cells. Suitablemedia for culturing E. coli cells include for example, Luria Broth (LB)and/or Terrific Broth (TB). Suitable media for culturing eukaryoticcells are Rosewell Park Memorial Media 1640 (RPMI 1640), MinimalEssential Media (MEM), Dulbecco's Modified Eagles Media (DMEM), all ofwhich may be supplemented with serum and/or growth factors as indicatedby the particular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate and/or fetal calf serum as necessary.

[0215] Typically, an antibiotic or other compound useful for selectivegrowth of transformed cells is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin. Othercompounds for selctive growth media include ampicillin, tetracycline andneomycin. The amount of Fhm polypeptide produced by a host cell can beevaluated using standard methods known in the art. Such methods include,without limitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays such as DNA binding gelshift assays.

[0216] If a Fhm polypeptide has been designed to be secreted from thehost cells, the majority of polypeptide may be found in the cell culturemedium. If however, the Fhm polypeptide is not secreted from the hostcells, it will be present in the cytoplasm and/or nucleus (foreukaryotic host cells) or in the cytosol (bacterial host cells).

[0217] The intracellular material (including inclusion bodies forgram-negative bacteria) can be extracted from the host cell using anystandard technique known to the skilled artisan. For example, the hostcells can be lysed to release the contents of the periplasm/cytoplasm byFrench press, homogenization, and/or sonication followed bycentrifugation.

[0218] If a Fhm polypeptide has formed inclusion bodies in the cytosol,the inclusion bodies can often bind to the inner and/or outer cellularmembranes and thus will be found primarily in the pellet material aftercentrifugation. The pellet material can then be treated at pH extremesor with a chaotropic agent such as a detergent, guanidine, guanidinederivatives, urea, or urea derivatives in the presence of a reducingagent such as dithiothreitol at alkaline pH or tris carboxyethylphosphine at acid pH to release, break apart, and solubilize theinclusion bodies. The Fhm polypeptide in its now soluble form can thenbe analyzed using gel electrophoresis, immunoprecipitation or the like.If it is desired to isolate the Fhm polypeptide, isolation may beaccomplished using standard methods such as those described herein andin Marston et al. (Meth. Enz., 182:264-275 1990).

[0219] In some cases, a Fhm polypeptide may not be biologically activeupon isolation. Various methods for “refolding” or converting thepolypeptide to its tertiary structure and generating disulfide linkages,can be used to restore biological activity. Such methods includeexposing the solubilized polypeptide to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization, but usually the chaotrope is used at a lowerconcentration and is not necessarily the same as chaotropes used for thesolubilization. In most cases the refolding/oxidation solution will alsocontain a reducing agent or the reducing agent plus its oxidized form ina specific ratio to generate a particular redox potential allowing fordisulfide shuffling to occur in the formation of the protein's cysteinebridge(s). Some of the commonly used redox couples includecysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, 2-mercaptoethanol(βME)/dithio-β(ME). Acosolvent is necessary to increase the efficiency of the refolding, andthe more common reagents used for this purpose include glycerol,polyethylene glycol of various molecular weights, arginine and the like.

[0220] If inclusion bodies are not formed to a significant degree uponexpression of a Fhm polypeptide, then the polypeptide will be foundprimarily in the supernatant after centrifugation of the cellhomogenate. The polypeptide and may be further isolated from thesupernatant using methods such as those described herein.

[0221] The purification of an Fhm polypeptide from solution can beaccomplished using a variety of techniques. If the polypeptide has beensynthesized such that it contains a tag such as Hexahistidine (Fhmpolypeptide/hexaHis) or other small peptide such as FLAG (Eastman KodakCo., New Haven, Conn.) or myc (Invitrogen, Carlsbad, Calif.) at eitherits carboxyl or amino terminus, it may be purified in a one-step processby passing the solution-through an affinity column where the columnmatrix has a high affinity for the tag.

[0222] For example, polyhistidine binds with great affinity andspecificity to nickel, thus annickel; thus affinity column of nickel(such as the Qiagen® nickel columns) can be used for purification of Fhmpolypeptide/polyHis. See for example, Ausubel et al., eds., CurrentProtocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, NewYork 1993.

[0223] Additionally, the Fhm polypeptide may be purified through the useof a monoclonal antibody which is capable of specifically recognizingand binding to the Fhm polypeptide.

[0224] Suitable procedures for purification thus include, withoutlimitation, affinity chromatography, immunoaffinity chromatography, ionexchange chromatography, molecular sieve chromatography, HighPerformance Liquid Chromatography (HPLC), electrophoresis (includingnative gel electrophoresis) followed by gel elution, and preparativeisoelectric focusing (“Isoprime” machine/technique, Hoefer Scientific,San Francisco, Calif.). In some cases, two or more purificationtechniques may be combined to achieve increased purity.

[0225] Fhm polypeptides, fragments, and/or derivatives thereof may alsobe prepared by chemical synthesis methods (such as solid phase peptidesynthesis) using techniques known in the art, such as those set forth byMerrifield et al., (J. Am. Chem. Soc., 85:2149, 1963), Houghten et al.(Proc Natl Acad. Sci. USA, 82:5132, 1985), and Stewart and Young (SolidPhase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill., 1984).Such polypeptides may be synthesized with or without a methionine on theamino terminus. Chemically synthesized Fhm polypeptides or fragments maybe oxidized using methods set forth in these references to formdisulfide bridges. Chemically synthesized Fhm polypeptides, fragments orderivatives are expected to have comparable biological activity to thecorresponding Fhm polypeptides, fragments or derivatives producedrecombinantly or purified from natural sources, and thus may be usedinterchangeably with recombinant or natural Fhm polypeptide.

[0226] Another means of obtaining Fhm polypeptide is via purificationfrom biological samples such as source tissues and/or fluids in whichthe Fhm polypeptide is naturally found. Such purification can beconducted using methods for protein purification as described above. Thepresence of the Fhm polypeptide during purification may be monitored,for example, using an antibody prepared against recombinantly producedFhm polypeptide or peptide fragments thereof.

[0227] A number of additional methods for producing nucleic acids andpolypeptides are known in the art, and the methods can be used toproduce polypeptides having specificity for Fhm. See for example,Roberts et al., Proc. Natl. Acad. Sci. USA, 94:12297-12303, 1997, whichdescribes the production of fusion proteins between an mRNA and itsencoded peptide. See also Roberts, Curr. Opin. Chem. Biol., 3:268-273,1999.

[0228] Additionally, U.S. Pat. No. 5,824,469 describes methods ofobtaining oligonucleotides capable of carrying out a specific biologicalfunction. The procedure involves generating a heterogeneous pool ofoligonucleotides, each having a 5′ randomized sequence, a centralpreselected sequence, and a 3′ randomized sequence. The resultingheterogeneous pool is introduced into a population of cells that do notexhibit the desired biological function. Subpopulations of the cells arethen screened for those which exhibit a predetermined biologicalfunction. From that subpopulation, oligonucleotides capable of carryingout the desired biological function are isolated. U.S. Pat. Nos.5,763,192, 5,814,476, 5,723,323, and 5,817,483 describe processes forproducing peptides or polypeptides. This is done by producing stochasticgenes or fragments thereof, and then introducing these genes into hostcells which produce one or more proteins encoded by the stochasticgenes. The host cells are then screened to identify those clonesproducing peptides or polypeptides having the desired activity.

[0229] Another method for producing peptides or polypeptides isdescribed in PCT/US98/20094 (WO99/15650) filed by Athersys, Inc. Knownas “Random Activation of Gene Expression for Gene Discovery” (RAGE-GD),the process involves the activation of endogenous gene expression orover-expression of a gene by in situ recombination methods. For example,expression of an endogenous gene is activated or increased byintegrating a regulatory sequence into the target cell which is capableof activating expression of the gene by non-homologous or illegitimaterecombination. The target DNA is first subjected to radiation, and agenetic promoter inserted. The promoter eventually locates a break atthe front of a gene, initiating transcription of the gene. This resultsin expression of the desired peptide or polypeptide.

[0230] It will be appreciated that these methods can also be used tocreate comprehensive IL-17 like protein expression libraries, which cansubsequently be used for high throughput phenotypic screening in avariety of assays, such as biochemical assays. cellular assays, andwhole organism assays (e.g., plant, mouse, etc.).

[0231] Proteins, Polypeptides, Fragments, Variants and Muteins of Fhm:

[0232] Polypeptides of the invention include isolated Fhm polypeptidesand polypeptides related thereto including fragments, variants, fusionpolypeptides, and derivatives as defined hereinabove.

[0233] Fhm fragments of the invention may result from truncations at theamino tern-inus (with or without a leader sequence), truncations at thecarboxy terminus, and/or deletions internal to the polypeptide. Mostdeletions and insertions, and substitutions in particular, are notexpected to produce radical changes in the characteristics of the Fhmprotein. However, when it is difficult to predict the exact effect ofthe substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays. For example, a variant typically is made bysite-specific mutagenesis of the Fhm-encoding nucleic acid, expressionof the variant nucleic acid in recombinant cell culture, and,optionally, purification from the cell culture, for example, byimmunoaffinity adsorption on a polyclonal anti-Fhm antibody column (toabsorb the variant by binding it to at least one remaining immuneepitope). In preferred embodiments, truncations and/or deletionscomprise about 10 amino acids, or about 20 amino acids, or about 50amino acids, or about 75 amino acids, or about 100 amino acids, or morethan about 100 amino acids. The polypeptide fragments so produced willcomprise about 25 contiguous amino acids, or about 50 amino acids, orabout 75 amino acids, or about 100 amino acids, or about 150 aminoacids, or about 200 amino acids. Such Fhm polypeptides fragments mayoptionally comprise an amino terminal methionine residue.

[0234] Fhm polypeptide variants of the invention include one or moreamino acid substitutions, additions and/or deletions as compared to SEQID NO: 4. In preferred embodiments, the variants have from 1 to 3, orfrom 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or morethan 100 amino acid substitutions, insertions, additions and/ordeletions, wherein the substitutions may be conservative, as definedabove, or non-conservative or any combination thereof. Moreparticularly, Fhm variants may comprise the amino acid sequence set outas SEQ ID NO: 4, wherein one or more amino acids from the groupconsisting of amino acids 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176 up to 251 is substituted with another amino acid. Thevariants may have additions of amino acid residues either at the carboxyterminus or at the amino terminus (with or without a leader sequence).

[0235] Preferred Fhm polypeptide variants include glycosylation variantswherein the number and/or type of glycosylation sites has been alteredcompared to native Fhm polypeptide. In one embodiment, Fhm variantscomprise a greater or a lesser number of N-linked glycosylation sites. AN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Thr, where the amino acid residue designated as X may be any type ofamino acid except proline. Substitution(s) of amino acid residues tocreate this sequence provides a potential new site for addition of aN-linked carbohydrate chain. Alternatively, substitutions to eliminatethis sequence will remove an existing N-linked carbohydrate chain. Alsoprovided is a rearrangement of N-linked carbohydrate chains wherein oneor more N-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated.

[0236] One skilled in the art will be able to determine suitablevariants of the native Fhm polypeptide using well known techniques. Forexample, one may be able to predict suitable areas of the molecule thatmay be changed without destroying biological activity. Also, one skilledin the art will realize that even areas that may be important forbiological activity or for structure may be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the polypeptide structure.

[0237] For predicting suitable areas of the molecule that may be changedwithout destroying activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of Fhm polypeptide to such similar polypeptides. After makingsuch a comparison, one skilled in the art would be able to determineresidues and portions of the molecules that are conserved among similarpolypeptides. One skilled in the art would know that changes in areas ofthe Fhm molecule that are not conserved would be less likely toadversely affect biological activity and/or structure. One skilled inthe art would also know that, even in relatively conserved regions, onecould have likely substituted chemically similar amino acids for thenaturally occurring residues while retaining activity (e.g. conservativeamino acid residue substitutions).

[0238] Also, one skilled in the art may review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one skilled inthe art can predict the importance of amino acid residues in Fhm thatcorrespond to amino acid residues that are important for activity orstructure in similar polypeptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of Fhm.

[0239] If available, one skilled in the art can also analyze the crystalstructure and amino acid sequence in relation to that structure insimilar polypeptides. In view of that information, one skilled in theart may be able to predict the alignment of amino acid residues of Fhmpolypeptide with respect to its three dimensional structure. One skilledin the art may choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules.

[0240] Moreover, one skilled in the art can generate test variantscontaining a single amino acid-substitution at each amino acid residue.The variants can be screened using activity assays disclosed in thisapplication. Such variants are used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed activity, variants with such achange would be avoided. Thus, based on information gathered from suchexperiments, when attempting to find additional acceptable variants, oneskilled in the art can determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

[0241] Fhm polypeptide analogs of the invention can be determined bycomparing the amino acid sequence of Fhm polypeptide with related familymembers. Exemplary Fhm polypeptide related family members include, butare not limited to, the TNF-α, TNK-β, LyT-β, FasL, CD40L, CD30L, OPGL,and TRAIL. This comparison can be accomplished by using a Pileupalignment (Wisconsin GCG Program Package) or an equivalent (overlapping)comparison with multiple family members within conserved andnon-conserved regions.

[0242] As shown in FIG. 1, the predicted amino acid sequence of Fhmpolypeptide (SEQ ID NO: 4) is aligned with the corresponding regions ofhuman FasL, mouse FasL, rat FasL, human CD40L, mouse CD40L, mouse OPGL,human OPGL, human TRAIL, mouse TRAIL, human CD30L, human CD30L, humanLyT-β, mouse LyT-β, human TNF-β, mouse TNF-β, human TNF-α and mouseTNF-α. (SEQ ID NOS: 5-21). Other Fhm polypeptide analogs can bedetermined using these or other methods known to those of skill in theart. These overlapping sequences provide guidance for conservative andnon-conservative amino acids substitutions resulting in additional Fhmanalogs. It will be appreciated that these amino acid substitutions canconsist of naturally occurring or non-naturally occurring amino acids.For example, as depicted in FIG. 1, alignment of the B/B′ loop and D/Eloop of these ligands indicates potential Fhm analogs may have the Valresidue at position 153 substituted with a Ile, Met, Leu, Phe, Ala orNorleucine residue, the Tyr residue at position 147 may be substitutedwith, or the Phe residue at position 154 may be substituted with Leu,Val, Ile, Ala, or Tyr residue. Further, the Ser residue at position 151may be substituted with Thr, Ala, or Cys, the Gly residue at 145 may besubstituted with Pro or Ala, and the Tyr at position 150 may besubstituted with Trp, Phe, Thr or Ser.

[0243] Fhm fusion polypeptides of the invention comprise Fhmpolypeptides, fragments, variants, or derivatives fused to one or moreheterologous peptides or proteins. Heterologous peptides and proteinsinclude, but are not limited to, an epitope to allow for detectionand/or isolation of a Fhm fusion polypeptide, a transmembrane receptorprotein or a portion thereof, such as an extracellular domain, or atransmembrane, a ligand or a portion thereof which binds to atransmembrane receptor protein, an enzyme or portion thereof which iscatalytically active, a protein or peptide which promotesoligomerization, such as leucine zipper domain, and a protein or peptidewhich increase stability, such as an immunoglobulin constant region. AFhm polypeptide may be fused to itself or to a fragment, variant, orderivative thereof. Fusions may be made either at the amino terminus orat the carboxy terminus of a Fhm polypeptide, and may be direct with nolinker or adapter molecule or may be through a linker or adaptermolecule, such as one or more amino acid residues up to about 20 aminoacids residues, or up to about 50 amino acid residues. Alternatively,the Fhm fusion protein may comprise one or two Fhm polypeptidescovalently linked to one or two TNF ligand polypeptide(s), or a memberof the TNF ligand family or a cytokine receptor such as interleukin-1(IL-1) polypeptide. The ligands preferably are produced as fusionproteins using recombinant DNA technology. A linker or adapter moleculemay also be designed with a cleavage site for a DNA restrictionendonuclease or for proteolytic cleavage to allow for separation andsubsequent folding of the fused moieties.

[0244] Also envisioned as a part of the invention are circularlypermuted structural analogs of the Fhm polypeptide.

[0245] The development of recombinant DNA methods has made it possibleto study the effects of sequence transposition on protein folding,structure and function. The approach used in creating new sequencesresembles that of naturally occurring pairs of proteins that are relatedby linear reorganization of their amino acid sequences (Cunningham, etal., Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222, 1979; Teather & Ernie,J. Bacteriol. 172:3837-3841, 1990; Schimming et al., Eur. J. Biochem.204:13-19,1992; Yamiuchi and Minamikawa, FEBS Lett 260:127-130,1991;MacGregor et al., FEBS Lett. 378:263-266, 1996). The first in vitroapplication of this type of rearrangement to proteins was described byGoldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). A newN-terminus is selected at an internal site (breakpoint) of the originalsequence, the new sequence having the same order of amino acids as theoriginal from the breakpoint until it reaches an amino acid that is ator near the original C-terminus. At this point the new sequence isjoined, either directly or through an additional portion of sequence(linker), to an amino acid that is at or near the original N-terminus,and the new sequence continues with the same sequence as the originaluntil it reaches a point that is at or near the amino acid that wasN-terminal to the breakpoint site of the original sequence, this residueforming the new C-terminus of the chain.

[0246] This approach has been applied to proteins which range in sizefrom 58 to 462 amino acids (Goldenberg & Creighton, J. Mol. Biol.165:407-413, 1983; Li & Coffino, Mol. Cell. Biol. 13:2377-2383, 1993).The proteins examined have represented a broad range of structuralclasses, including proteins that contain predominantly α-helix(interleukin-4; Kreitman et al., Cytokine 7:311-318, 1995),predominantly β-sheet (interleukin-1; Horlick et al., Protein Eng.5:427-431, 1992), or mixtures of the two (yeast phosphoribosylanthranilate isomerase; Luger et al., Science 243:206-210, 1989).

[0247] In a preferred embodiment, a Fhm polypeptide, fragment, variantand/or derivative is fused to an Fc region of human IgG. In one example,a human IgG hinge, CH2 and CH3 region may be fused at either theN-terminus or C-terminus of the Fhm polypeptides using methods known tothe skilled artisan. In another example, a portion of a hinge regionsand CH2 and CH3 regions may be fuse. The Fhm Fc-fusion polypeptide soproduced may be purified by use of a Protein A affinity column (Pierce,Rockford, Ill.). In addition, peptide and proteins fused to an Fc regionhave been found to exhibit a substantially greater half-life in vivothan the unfused counterpart. Also, a fusion to an Fc region allows fordimerization/multimerization of the fusion polypeptide. The Fc regionmay be naturally occurring Fc region, or may be altered to improvecertain qualities such as therapeutic qualities, circulation time,reduce aggregation, etc.

[0248] Fhm polypeptide derivatives are also included in the scope of thepresent invention. Covalent modifications of the Fhm proteins of thepresent invention are included within the scope of this invention.Variant Fhm proteins may be conveniently prepared by in vitro synthesis.Such modifications may be introduced into the molecule by reactingtargeted amino acid residues of the purified or crude protein with anorganic derivatizing agent that is capable of reacting with selectedside chains or terminal residues. The resulting covalent derivatives areuseful in programs directed at identifying residues important forbiological activity.

[0249] Cysteinyl residues most commonly are reacted with α-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carbocyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophencl, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0250] Histidyl residues-are derivatized by reaction withdiethylprocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0.

[0251] Lysinyl and amino terminal residues are reacted with succinic orcarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylissurea; 2,4 pentanedione; and transaminase catalyzed reactionwith glyoxylate.

[0252] Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineEpsilon-amino group.

[0253] The specific modification of tyrosyl residues per se has beenstudied extensively, with particular interest in introducing spectrallabels into tyrosyl residues by reaction with aromatic diazoniumcompounds or tetranitromethane. Most commoly, N-acetylimidizol andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively. Tyrosyl residues are iodinated using ¹²⁵I or¹³¹I to prepare labeled proteins for use in radioimmunoassay, thechloramine T method described above being suitable.

[0254] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiumides (R¹) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carboduimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions.

[0255] Derivatization with bifunctional agents is useful forcrosslinking the Fhm protein(s)/polypeptide to water-insoluble supportmatrixes or surfaces for use in the method for cleaving the Fhmprotein-fusion polypeptide to release and recover the cleavedpolypeptide. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homo-bifunctional imidoesters, including disuccinimidyl esterssuch as 3,3′-dithiiobis(succinimidylpropioonate), and bifunctionalmaleimides such as bix-N-maleimido-1,8-octane. Derivatizing agents suchas methyl-3-[p-azidophenyl) dithio]propioimidate yield photoactivatableintermediates that are capable of forming cross links in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440, incorporated herein by reference, are employed forprotein immobilization.

[0256] Glutaminyl and asparaginyl residues are frequently deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese-residues falls within the scope of this invention.

[0257] Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or theonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W. H. Freeman & Co., San Francisco, pp. 79-86,1983),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups. Such derivatives are chemicallymodified Fhm polypeptide compositions in which Fhm polypeptide is linkedto a polymer. The polymer selected is typically water soluble so thatthe protein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. The polymer selectedis usually modified to have a single reactive group, such as an activeester for acylation or an aldehyde for alkylation, so that the degree ofpolymerization may be controlled as provided for in the present methods.The polymer may be of any molecular weight, and may be branched orunbranched. Included within the scope of the Fhm polypeptide polymers isa mixture of polymers. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable.

[0258] The polymers each may be of any molecular weight and may bebranched or unbranched. The polymers each typically have an averagemolecular weight of between about 2 k kDa to about 100 kDa (the term“about” indicating that in preparations of a water soluble polymer, somemolecules will weigh more, some less, than the stated molecular weight).The average molecular weight of each polymer is between about 5 kDa and5 kDa, about 50 kDa, more preferably between about 12 kDa to about 40kDa and most preferably between about 20 kDa to about 35 kDa.

[0259] Suitable water soluble polymers or mixtures thereof include, butare not limited to, N-linked or O-linked carbohydrates, sugars,phosphates, carbohydrates; sugars; phosphates; polyethylene glycol (PEG)(including the forms of PEG that have been used to derivatize proteins,including mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol);monomethoxy-polyethylene glycol; dextran (such as low molecular weightdextran, of, for example about 6 kD), cellulose; cellulose; othercarbohydrate-based polymers, poly-(N-vinyl pyrrolidone) polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol. Also encompassed by the present invention arebifunctional crosslinking molecules which may be used to preparecovalently attached multimers of the polypeptide comprising the aminoacid sequence of SEQ ID NO: 4 or an Fhm polypeptide variant.

[0260] In general, chemical derivatization may be performed under anysuitable condition used to react a protein with an activated polymermolecule. Methods for preparing chemical derivatives of polypeptideswill generally comprise the steps of (a) reacting the polypeptide withthe activated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby thepolypeptide comprising the amino acid sequence of SEQ ID NO: 4, or anFhm polpeptide variant becomes attached to one or more polymermolecules, and (b) obtaining the reaction product(s). The optimalreaction conditions will be determined based on known parameters and thedesired result. For example, the larger the ratio of polymer molecules:protein, the greater the percentage of attached polymer molecule. In oneembodiment, the Fhm polypeptide derivative may have a single polymermolecule moiety at the amino terminus. (See, e.g., U.S. Pat. No.5,234,784).

[0261] A particularly preferred water-soluble polymer for use herein ispolyethylene glycol, abbreviated PEG. As used herein, polyethyleneglycol is meant to encompass any of the forms of PEG that have been usedto derivatize other proteins, such as mono-(C1-C10) alkoxy- oraryloxy-polyethylene glycol. PEG is a linear or branched neutralpolyether, available in a broad range of molecular weights, and issoluble in water and most organic solvants. PEG is effective atexcluding other polymers or peptides when present in water, primarilythrough its high dynamic chain mobility and hydrophibic nature, thuscreating a water shell or hydration sphere when attached to otherproteins or polymer surfaces. PEG is nontoxic, non-immunogenic, andapproved by the Food and Drug Administration for internal consumption.

[0262] Proteins or enzymes when conjugated to PEG have demonstratedbioactivity, non-antigenic properties, and decreased clearance rateswhen administered in animals. F. M. Veronese et al., Preparation andProperties of Monomethoxypoly(ethylene glyco.)-modified Enzymes forTherapeutic Applications, in J. M. Harris ed., Poly(Ethylene Clycol)Chemistry—Biotechnical and Biomedical Applications 127-36, 1992,incorporated herein by reference. This is due to the exclusionproperties of PEG in preventing recognition by the immune system. Inaddition, PEG has been widely used in surface modification procedures todecrease protein adsorption and improve blood compatibility. S. W. Kimet al., Ann. N.Y. Acad. Sci. 516: 116-30 1987; Jacobs et al., Artif.Organs 12: 500-501, 1988; Park et al., J. Poly. Sci, Part A 29:1725-31,1991, incorporated herein by reference. Hydrophobic polymer surfaces,such as polyurethanes and polystyrene were modified by the grafting ofPEG (MW 3,400) and employed as nonthrombogenic surfaces. In thesestudies, surface properties (contact angle) were more consistent withhydrophilic surfaces due to the hydrating effect of PEG. Moreimportantly, protein (albumin and other plasma proteins) adsorption wasgreatly reduced, resulting from the high chain motility hydrationsphere, and protein exclusion properties of PEG.

[0263] PEG (MW 3,4000) was determined as an optimal size in surfaceimmobilization studies, Park et al., J. Biomed. Mat. Res. 26:739-45,1992, while PEG (MW 5,000) was most beneficial in decreasing proteinantigenicity. (F. M. Veronese et al., In J. M. Harris et., Poly(EthyleneGlycol) Chemistry—Biotechnical and Biomedical Applications 127-36,supra., incorporated herein by reference)

[0264] In general, chemical derivatization may be performed under anysuitable conditions used to react a biologically active substance withan activated polymer molecule. Methods for preparing pegylated Fhmpolypeptides will generally comprise the steps of (a) reacting thepolypeptide with polyethylene glycol (such as a reactive ester oraldehyde derivative of PEG) under conditions whereby Fhm polypeptidebecomes attached to one or more PEG groups, and (b) obtaining thereaction product(s). In general, the optimal reaction conditions for theacylation reactions will be determined based on known parameters and thedesired result. For example, the larger the ratio of PEG: protein, thegreater the percentage of poly-pegylated product.

[0265] In a preferred embodiment, the Fhm polypeptide derivative willhave a single PEG moiety at the N terminus. See U.S. Pat. No. 8,234,784,herein incorporated by reference.

[0266] Generally, conditions which may be alleviated or modulated byadministration of the present Fhm polypeptide derivative include thosedescribed herein for Fhm polypeptides. However, the Fhm polypeptidederivative disclosed herein may have additional activities, enhanced orreduced biological activity, or other characteristics, such as increasedor decreased half-life, as compared to the non-derivatized molecules.

[0267] Genetically Engineered Non-Human Animals

[0268] Additionally included within the scope of the present inventionare non-human animals such as mice, rats, or other rodents, rabbits,goats, or sheep, or other farm animals, in which the gene (or genes)encoding the native Fhm polypeptide has (have) been disrupted (“knockedout”) such that the level of expression of this gene or genes is(are)significantly decreased or completely abolished. Such animals may beprepared using techniques and methods such as those described in U.S.Pat. No. 5,557,032.

[0269] The present invention further includes non-human animals such asmice, rats, or other rodents, rabbits, goats, sheep, or other farmanimals, in which either the native form of the Fhm gene(s) for thatanimal or a heterologous Fhm gene(s) is (are) over-expressed by theanimal, thereby creating a “transgenic” animal. Such transgenic animalsmay be prepared using well knownwell-known methods such as thosedescribed in U.S. Pat. No. 5,489,743 and PCT application No. WO94/28122.Application No. WO 94/28122.

[0270] The present invention further includes non-human animals in whichthe promoter for one or more of the Fhm polypeptides of the presentinvention is either activated or inactivated (e.g., by using homologousrecombination methods) to alter the level of expression of one or moreof the native Fhm polypeptides.

[0271] These non-human animals may be used for drug candidate screening.In such screening, the impact of a drug candidate on the animal may bemeasured; for example, drug candidates may decrease or increase theexpression of the Fhm gene. In certain embodiments, the amount of Fhmpolypeptide, that is produced may be measured after the exposure of theanimal to the drug candidate. Additionally, in certain embodiments, onemay detect the actual impact of the drug candidate on the animal. Forexample, the overexpression of a particular gene may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease expression of the geneor its ability to prevent or inhibit a pathological condition. In otherexamples, the production of a particular metabolic product such as afragment of a polypeptide, may result in, or be associated with, adisease or pathological condition. In such cases, one may test a drugcandidate's ability to decrease the production of such a metabolicproduct or its ability to prevent or inhibit a pathological condition.

[0272] Microarray

[0273] It will be appreciated that DNA microarray technology can beutilized in accordance with the present invention. DNA microarrays areminiature, high density arrays of nucleic acids positioned on a solidsupport, such as glass. Each cell or element within the array hasnumerous copies of a single species of DNA which acts as a target forhybridization for its cognate mRNA. In expression profiling using DNAmicroarray technology, mRNA is first extracted from a cell or tissuesample and then converted enzymatically to fluorescently labeled cDNA.This material is hybridized to the microarray and unbound cDNA isremoved by washing. The expression of discrete genes represented on thearray is then visualized by quantitating the amount of labeled cDNAwhich is specifically bound to each target DNA. In this way, theexpression of thousands of genes can be quantitated in a highthroughput, parallel manner from a single sample of biological material.

[0274] This high throughput expression profiling has a broad range ofapplications with respect to the Fhm molecules of the invention,including, but not limited to: the identification and validation of Fhmdisease-related genes as targets for therapeutics; molecular toxicologyof Fhm molecules and inhibitors thereof; stratification of populationsand generation of surrogate markers for clinical trials; and enhancingFhm-related small molecule drug discovery by aiding in theidentification of selective compounds in high throughput screens (HTS).

[0275] Selective Binding Agents

[0276] As used herein, the term “selective binding agent” refers to amolecule which has specificity for one or more Fhm polypeptides.Suitable selective binding agents include, but are not limited to,antibodies and derivatives thereof, polypeptides, and small molecules.Suitable selective binding agents may be prepared using methods known inthe art. An exemplary Fhm polypeptide selective binding agent of thepresent invention is capable of binding a certain portion of the Fhmpolypeptide thereby inhibiting the binding of the polypeptide to the Fhmpolypeptide receptor(s).

[0277] Selective binding agents such as antibodies and antibodyfragments that bind Fhm polypeptides are within the scope of the presentinvention. The antibodies may be polyclonal including monospecificpolyclonal, monoclonal (mAbs), recombinant, chimeric, humanized such asCDR-grafted, human, single chain, and/or bispecific, as well asfragments, variants or derivatives thereof. Antibody fragments includethose portions of the antibody which bind to an epitope on the Fhmpolypeptide. Examples of such fragments include Fab and F(ab′) fragmentsgenerated by enzymatic cleavage of full-length antibodies. Other bindingfragments include those generated by recombinant DNA techniques, such asthe expression of recombinant plasmids containing nucleic acid sequencesencoding antibody variable regions.

[0278] Polyclonal antibodies directed toward a Fhm polypeptide generallyare produce in animals (e.g. rabbits or mice) by means of multiplesubcutaneous or intraperitoneal injections of Fhm and an adjuvant. Itmay be useful to conjugate a Fhm polypeptide, or a variant, fragment orderivative thereof to a carrier protein that is immunogenic in thespecies to be immunized, such as keyhole limpet heocyanin, serum,albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also,aggregating agents such as alum are used to enhance the immune response.After immunization, the animals are bled and the serum is assayed foranti-Fhm antibody titer.

[0279] Monoclonal antibodies directed toward Fhm are produced using anymethod which provides for the production of antibody molecules bycontinuous cell lines in culture.

[0280] Examples of suitable methods for preparing monoclonal antibodiesinclude the hybridoma method of Kohler et al., Nature 256: 495-497,1975, and the human B-cell hybridoma method, Kozbor, J. Immunol. 133:3001, 1984; Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987).

[0281] Also provided by the invention are hybridoma cell lines whichproduce monoclonal antibodies reactive with Fhm polypeptides.

[0282] Monoclonal antibodies of the invention may be modified for use astherapeutics. One embodiment is a “chimeric” antibody in which a portionof the heavy and/or light chain is identical with or homologous tocorresponding sequence in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is/are identical with or a homologous tocorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. Also included arefragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; Morrison, et al.,Proc. Natl. Acad. Sci. U.S.A. 81: 6851-6855, 1985; incorporated hereinby reference).

[0283] In another embodiment, a monoclonal antibody of the invention isa “humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art (see U.S. Pat. Nos. 5,585,089 and 5,693,762).Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. Humanization can beperformed, for example, methods described in the art (Jones et al.,Nature 321: 522-525, 1986; Riechmann et al., Nature, 332: 323-327, 1988;Verhoeyen et al., Science 239: 1534-1536, 1988), by substituting atleast a portion of a rodent complementarity-determining region (CDR) forthe corresponding regions of a human antibody.

[0284] Also encompassed by the invention are fully human antibodieswhich bind Fhm polypeptides, fragments, variants and/or derivatives.Such antibodies are produced by immunization with a Fhm antigenoptionally conjugated to a carrier (i.e., at least having 6 contiguousamino acids). Using transgenic animals (e.g., mice) that are capable ofproducing a repertoire of human antibodies in the absence of endogenousimmunoglobulin production. See, for example, Jakobovits, et al., Proc.Natl. Acad. Sci. U.S.A. 90: 2551-2555, 1993; Jakobovits, et al., Nature362: 255-258, 1993; Bruggermann, et al., Year in Immuno. 7:33, 1993. Inone method, such transgenic animals are produced by incapacitating theendogenous loci encoding the heavy and light immunoglobulin chainstherein, and inserting loci encoding human heavy and light chainproteins into the genome thereof. Partially modified animals, that isthose having less than the full complement of modifications, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies with human (rather than e.g., murine) amino acidsequences, including variable regions which are immunospecific for theseantigens. See PCT Application Nos. PCT/US96/05928 and PCT/US93/06926.Additional methods are described in U.S. Pat. No. 5,545,807, PCTapplication nos. PCT/US91/245, PCT/GB89/01207, and in EP 546073B1 and EP546073A1. Human antibodies may also be produced by the expression ofrecombinant DNA in host cells or by expression in hybridoma cells asdescribed herein.

[0285] In an alternative embodiment, human antibodies can be produced inphage-display libraries (Hoogenboom, et al., J. Mol. Biol. 227:381,1991; Marks. et al. J. Mol. Biol. 222:581, 1991. These processes mimicimmune selection through the display of antibody repertoires on thesurface of filamentous bacteriophage, and subsequent selection of phageby their binding to an antigen of choice. One such technique isdescribed in PCT Application No. PCT/US98/17364, which describes theisolation of high affinity and functional agonistic antibodies for MPL-and msk-receptors using such an approach.

[0286] Chimeric, CDR grafted, and humanized antibodies are typicallyproduced by recombinant methods. Nucleic acids encoding the antibodiesare introduced into host cells and expressed using materials andprocedures described herein. In a preferred embodiment, the antibodiesare produced in mammalian host cells, such as CHO cells. Monoclonal(e.g., human) antibodies may be produced by the expression ofrecombinant DNA in host cells or by expression in hybridoma cells asdescribed herein.

[0287] The anti-Fhm antibodies of the invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays (Sola,Monoclonal Anitibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc., 1987)) for the detection and quantitation of Fhm polypeptides. Theantibodies will bind Fhm polypeptides with an affinity which isappropriate for the assay method being employed.

[0288] For diagnostic applications, in certain embodiments anti-Fhmantibodies typically may be labeled with a detectable moiety. Thedetectable moiety can be any one which is capable of producing, eitherdirectly or indirectly, a detectable signal. For example, the detectablemoiety may be a radioisotope, such as ³H, ¹⁴C, ³²p, 35w, or 125I, afluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, β-galactosidase, or horseradish peroxidase. See Bayer, etal., Meth. Enz. 184: 138-163, 1990.

[0289] The anti-Fhm antibodies of the invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays (Sola,Monoclonal Anitibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc., 1987)) for detection and quantitation of Fhm polypeptides. Theantibodies will bind Fhm polypeptides with an affinity which isappropriate for the assay method being employed.

[0290] The activity of the cell lysate or purified Fhm protein variantis then screened in a suitable screening assay for the desiredcharacteristic. For example, a change in the binding affinity for aligand or immunological character of the Fhm protein, such as affinityfor a given antibody, is measured by a competitive type immunoassay.Changes in immunomodulation activity are measured by the appropriateassay. Modifications of such protein properties as redox or thermalstability hydrophobicity, susceptibility to proteolytic degradation orthe tendency to aggregate with carriers or into multimers are assayed bymethods well known to the ordinarily skilled artisan. Competitivebinding assays rely on the ability of a labeled standard (e.g., a Fhmpolypeptide, or an immunologically reactive portion thereof) to competewith the test sample analyte (a Fhm polypeptide) for binding with alimited amount of antibody. The amount of a Fhm polypeptide in the testsample is inversely proportional to the amount of standard that becomesbound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies typically are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

[0291] Sandwich imuno-assays typically involve the use of twoantibodies, each capable of binding to a different immunogenic portion,or epitope, of the protein to be detected and/or quantitated. In asandwich assay, the test sample analyte typically is bound by a firstantibody which is immobilized on a solid support, and thereafter asecond antibody binds to the analyte, thus forming an insolublethree-part complex. See e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assays). Forexample, one type of sandwich assay is an enzyme linked immunosorbantassay (ELISA), in which case the detectable moiety is an enzyme.

[0292] The selective binding agents, including anti-Fhm antibodies arealso useful for in vivo imaging. An antibody labeled with a detectablemoiety may be administered to an animal, preferably into thebloodstream, and the presence and location of the labeled antibody inthe host is assayed. The antibody may be labeled with any moiety that isdetectable in an animal, whether by nuclear magnetic resonance,radiology, or other detection means known in the art.

[0293] Selective binding agents, including antibodies of the invention,may be used as therapeutics. These therapeutic antibodies are generallyagonists or antagonists, in that they either enhance or reduce,respectively, at least one of the biological activities of a Fhmpolypeptide. In one embodiment, antagonist antibodies of the inventionare antibodies or binding fragments thereof which are capable ofspecifically binding to a Fhm polypeptide, fragment, variant and/orderivative, and which are capable of inhibiting or eliminating thefunctional activity of a Fhm polypeptide in vivo or in vitro. Inpreferred embodiments, an antagonist antibody will inhibit thefunctional activity of a Fhm polypeptide at least about 50%, preferablyat least about 80%, more preferably 90%, and most preferably 100%. Inanother embodiment, the selective binding agent may be an antibody thatis capable of interacting with an Fhm binding partner (e.g., receptor)thereby inhibiting or eliminating Fhm activity in vitro or in vivo.Selective binding agents, including agonist and antagonist anti-Fhmantibodies, are identified by screening assays which are well known inthe art.

[0294] The invention also relates to a kit comprising Fhm selectivebinding agents (such as antibodies) and other reagents useful fordetecting Fhm polypeptide levels in biological samples. Such reagentsmay include, a detectable label, blocking serum, positive and negativecontrol samples, and detection reagents.

[0295] The Fhm polypeptides of the present invention can be used toclone Fhm receptors, using an expression cloning strategy. Radiolabeled(¹²⁵Iodine) Fhm polypeptide or affinity/activity-tagged Fhm polypeptide(such as an Fc fusion or an alkaline phosphatase fusion) can be used inbinding assays to identify a cell type or cell line or tissue thatexpresses Fhm receptor(s). RNA isolated from such cells or tissues canbe converted to cDNA, cloned into a mammalian expression vector andtransfected into mammalian cells (such-as COS or 293 cells) to create anexpression library. A radiolabeled or tagged Fhm polypeptide can then beused as an affinity ligand to identify and isolate from this library thesubset of cells which express the Fhm receptor(s) on their surface. DNAcan then be isolated from these cells and transfected into mammaliancells to create a secondary expression library in which the fraction ofcells expressing Fhm receptor(s) is many-fold higher than in theoriginal library. This enrichment process can be repeated iterativelyuntil a single recombinant clone containing an Fhm receptor is isolated.Isolation of the Fhm receptor(s) is useful for identifying or developingnovel agonists and antagonists of the Fhm polypeptide signaling pathway.Such agonists and antagonists include soluble Fhm receptor(s), anti-Fhmreceptor antibodies, small molecules, or antisense oligonucleotides, andthey may be used for treating, preventing, or diagnosing one or moredisease or disorder, including those described herein.

[0296] Diagnostic Kits and Reagents

[0297] This invention also contemplates use of Fhm proteins, fragmentsthereof, peptides, binding compositions, and their fusion products in avariety of diagnostic kits and methods for detecting the presence ofreceptors and/or antibodies. Typically the kit will have a compartmentcontaining a Fhm peptide or gene segment or a reagent which recognizesone or the other, e.g., binding reagents.

[0298] A kit for determining the binding affinity of a binding partneror a test compound to the Fhm would typically comprise a binding partnertest compound; a labeled compound, for example an antibody having knownbinding affinity for the protein; or a source of binding partner(naturally occurring or recombinant), and a means for separating boundfrom free labeled compound, such as a solid phase for immobilizing theligand or its binding partner. Once compounds are screened, those havingsuitable binding affinity to the ligand or its binding partner can beevaluated in suitable biological assays, as are well known in the art,to determine whether they act as agonists or antagonists of Fhmactivity.

[0299] The availability of recombinant Fhm and/or receptor polypeptidesalso provide well defined standards for calibrating such assays or aspositive control samples.

[0300] A preferred kit for determining the concentration of, forexample. Fhm-ligand and/or its cognate binding partner in a sample wouldtypically comprise a labeled compound, e.g., antibody, having knownbinding affinity for the target a source of ligand or receptor(naturally occurring or recombinant), and a means for separating thebound from free labeled compound, for example, a solid phase forimmobilizing the ligand or receptor. Compartments containing reagents,and instructions for use or disposal, will normally be provided.

[0301] Antibodies, including antigen binding fragments, specific for theligand or receptor, or fragments are useful in diagnostic applicationsto detect the presence of elevated levels of ligand, receptor, and/orits fragments. Such diagnostic assays can employ lysates, live cells,fixed cells, immunofluorescence, cell cultures, body fluids, and furthercan involve the detection of antigens related to the ligand or receptorin serum, or the like. Diagnostic assays may be homogeneous (without aseparation step between free reagent and antigen complex) orheterogeneous (with a separation step). Various commercial assays exist,such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay(ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassaytechnique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), andthe like. For example, unlabeled antibodies can be employed by using asecond antibody which is labeled and which recognizes the primaryantibody to a ligand or receptor or to a particular fragment thereof.Similar assays have also been extensively discussed in the literature.(See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press.)

[0302] Anti-idiotypic antibodies may have similar uses to diagnosepresence of antibodies against a ligand or receptor, as such may bediagnostic of various abnormal states. For example, overproduction of aligand or receptor may result in production of various immunologicalreactions which may be diagnostic of abnormal physiological states,particularly in various inflammatory or allergic conditions.

[0303] Frequently, the reagents for diagnostic assays are supplied inkits, so as to optimize the sensitivity of the assay. For the subjectinvention, depending upon the nature of the assay, the protocol, and thelabel, either labeled or unlabeled antibody or labeled ligand orreceptor is provided. This is usually in conjunction with otheradditives, such as buffers, stabilizers, materials necessary for signalproduction such as substrates for enzymes, and the like. Preferably, thekit will also contain instructions for proper use and disposal of thecontents after use. Typically the kit has compartments or containers foreach useful reagent. Desirably, the reagents are provided as a drylyophilized powder, where the reagents may be reconstituted in anaqueous medium providing appropriate concentrations of reagents forperforming the assay.

[0304] The aforementioned constituents of the drug screening and thediagnostic assays may be used without modification or may be modified ina variety of ways. For example, labeling may be achieved by covalentlyor non-covalently joining a moiety which directly or indirectly providesa detectable signal. In any of these assays, the ligand, test compound,receptor, or antibodies thereto can be labeled either directly orindirectly. Possibilities for direct labeling include label groups:radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) such asperoxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat.No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups.

[0305] There are also numerous methods of separating bound from the freeligand, or alternatively bound from free test compound. The ligand orreceptor can be immobilized on various matrixes, perhaps with detergentsor associated lipids, followed by washing. Suitable matrixes includeplastic such as an ELISA plate, filters, and beads. Methods ofimmobilizing the ligand or receptor to a matrix include, withoutlimitation, direct adhesion to plastic, use of a capture antibody,chemical coupling, and biotin-avidin. The last step in this approach mayinvolve the precipitation of antigen/antibody complex by any of severalmethods including those utilizing, e.g., an organic solvent such aspolyethylene glycol or a salt such as ammonium sulfate. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle et al. Clin.Chem., 30:1457-1461, 1984, and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,6178, incorporated hereinby reference.

[0306] Methods for linking proteins or their fragments to the variouslabels have been extensively reported in the literature and do notrequire detailed discussion here. Many of the techniques involve the useof activated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

[0307] Nucleic acid molecules of the invention may be used to map thelocations of the Fhm gene and related genes on chromosomes. Mapping maybe done by techniques known in the art, such as PCR amplification, insitu hybridization, and FISH.

[0308] This invention is also related to the use of the Fhm gene as partof a diagnostic assay for detecting diseases or susceptibility todiseases related to the presence of mutated Fhm gene. Such diseases arerelated to an abnormal expression of Fhm, for example, abnormal cellularproliferation such as tumors and cancers.

[0309] Individuals carrying mutations in the human Fhm gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324:163-166, 1986) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding Fhm polypeptide canbe used to identify and analyze Fhm mutations. For example, deletionsand insertions can be detected by a change in size of the amplifiedproduct in comparison to the normal genotype. Point mutations can beidentified by hybridizing amplified DNA to radiolabeled Fhm RNA oralternatively radiolabeled Fhm antisense DNA sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNaseA digestion or by differences in melting temperatures.

[0310] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturing,formamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242, 1985).

[0311] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci.,USA, 85:4397-4401, 1985).

[0312] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0313] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0314] The present invention also relates to a diagnostic assay fordetecting altered levels of Fhm protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease, for example, tumors, cerebral malaria and hereditary periodicfever syndromes. Assays used to detect levels of Fhm protein in a samplederived from a host are well-known to those of skill in the art andinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis, ELISA assays and “sandwich” assay. An ELISA assay (Coligan, etal., Current Protocols in Immunology, 1(2), Chapter 6, 1991) partiallycomprises preparing an antibody specific to the Fhm antigen, preferablya monoclonal antibody. In addition a reporter antibody is preparedagainst the monoclonal antibody. To the reporter antibody is attached adetectable reagent such as radioactivity, fluorescence or in thisexample a horseradish peroxidase enzyme. A sample is now removed from ahost and incubated on a solid support, e.g., a polystyrene dish, thatbinds the proteins in the sample. Any free protein binding sites on thedish are then covered by incubating with a non-specific protein likebovine serum albumin (BSA). Next, the monoclonal antibody is incubatedin the dish during which time the monoclonal antibodies attach to anyFhm proteins attached to the polystyrene dish. All unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is now placed in the dish resulting in binding ofthe reporter antibody to any monoclonal antibody bound to Fhm Unattachedreporter antibody is then washed out. Peroxidase substrates are thenadded to the dish and the amount of color developed in a given timeperiod is a measurement of the amount of Fhm protein present in a givenvolume of patient sample when compared against a standard curve.

[0315] A competition assay may be employed wherein antibodies specificto Fhm are attached to a solid support and labeled Fhm and a samplederived from the host are passed over the solid support and the amountof label detected, for example, by liquid scintillation chromotagraphy,can be correlated to a quantity of Fhm in the sample. In addition, asandwich immuno-assay as described above may also be carried out toquantify the amount of Fhm in a biological sample.

[0316] The sequences of the present invention are also valuable forchromosome identification and mapping. The sequence can be specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. Moreover, there is a current need foridentifying particular sites on the chromosome wherein a gene can belocalized. Few chromosome marking reagents based on actual sequence data(repeat polymorphisms) are presently available for marking chromosomallocation. The mapping of DNAs to chromosomes according to the presentinvention is an important first step in correlating those sequences withgenes associated with disease.

[0317] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the3′-untranslated region of the sequence is used to rapidly select primersthat do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers are then used forPCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

[0318] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map Fhm to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0319] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 500 or 600 bases; however, clones larger than 2,000 bp havea higher likelihood of binding to a unique chromosomal location withsufficient signal intensity for simple detection. FISH requires use ofgenomic clones or clones from which the express sequence tag (EST) wasderived, and the longer the better. For example, 2,000 bp is good, 4,000is better, and more than 4,000 is probably not necessary to get goodresults a reasonable percentage of the time. For a review of thistechnique see Verma et al., Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988).

[0320] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0321] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0322] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0323] The nucleic acid molecule(s) of the present invention are alsouseful as anti-sense inhibitors of Fhm expression. Such inhibition maybe effected by nucleic acid molecules which are complementary to andhybridize to expression control sequences (triple helix formation) or toFhm mRNA. Anti-sense probes may be designed by available techniquesusing the sequence of Fhm disclosed herein. Anti-sense inhibitorsprovide information relating to the decrease or absence of a Fhmpolypeptide in a cell or organism.

[0324] The nucleic acid molecules of the invention may be used for genetherapy. Nucleic acid molecules which express Fhm in vivo provideinformation relating to the effects of the polypeptide in cells ororganisms. Fhm nucleic acid molecules, fragments, and/or derivativesthat do not themselves encode biologically active polypeptides may beuseful as hybridization probes in diagnostic assays to test, eitherqualitatively or quantitatively, for the presence of Fhm DNA orcorresponding RNA in mammalian tissue or bodily fluid samples.

[0325] Fhm polypeptide fragments, variants, and/or derivatives, whetherbiologically active or not, are useful for preparing antibodies thatbind to an Fhm polypeptide. The antibodies may be used for in vivo andin vitro diagnostic purposes, such as in labeled form to detect thepresence of Fhm polypeptide in a body fluid or cell sample. Theantibodies may bind to an Fhm polypeptide so as to diminish or block atleast one activity characteristic of an Fhm polypeptide, or may bind toa polypeptide to increase an activity.

[0326] Assaying for Modulators of Fhm Polypeptide Activity:

[0327] In some situations, it may be desirable to identify moleculesthat are modulators, i.e., agonists or antagonists, of the activity ofFhm polypeptide. Natural or synthetic molecules that modulate Fhmpolypeptide can be identified using one or more of the screening assays,such as those described herein. Such molecules may be administeredeither in an ex vivo manner, or in an in vivo manner by local orintravenous (iv) injection, or by oral delivery, implantation device, orthe like.

[0328] “Test molecule(s)” refers to the molecule(s) that is/are underevaluation for the ability to modulate (i.e., increase or decrease) theactivity of an Fhm polypeptide. Most commonly, a test molecule willinteract directly with an Fhm polypeptide. However, it is alsocontemplated that a test molecule may also modulate Fhm polypeptideactivity indirectly, such as by affecting Fhm gene expression, or bybinding to an Fhm binding partner (e.g., receptor). In one embodiment, atest molecule will bind to an Fhm polypeptide with an affinity constantof at least about 10⁻⁶ M, preferably about 10 ⁻⁸ M, more preferablyabout 10⁻⁹ M, and even more preferably about 10⁻¹⁰ M.

[0329] Methods for identifying compounds which interact with Fhmreceptor polypeptides are encompassed by the invention. In certainembodiments, a Fhm receptor polypeptide is incubated with a testmolecule under conditions which permit interaction of the test moleculeto the receptor polypeptide, in the presence or absence of bioactiveFhm, and the extent of the interaction can be measured. The testmolecules can be screened in a substantially purified form or in a crudemixture.

[0330] In certain embodiments, a Fhm polypeptide agonist or antagonistmay be a protein, peptide, carbohydrate, lipid, or small molecularweight molecule which interacts with Fhm polypeptide to regulate itsactivity. Molecules which regulate Fhm polypeptide expression includenucleic acids which are complementary to nucleic acids encoding an Fhmpolypeptide, or are complementary to nucleic acids acid sequences whichdirect or control the expression of Fhm polypeptide, and which act asanti-sense regulators of expression.

[0331] The measurement of the interaction of test molecules withputative Fhm receptor polypeptide(s) in the presence or absence of Fhmligand may be carried out in several formats, including cell-basedbinding assays, membrane binding assays, solution-phase assays andimmunoassays. In general, test molecules are incubated with a putativeFhm receptor polypeptide for a specified period of time and Fhmpolypeptide activity is determined by one or more assays measuringbiological activity.

[0332] The interaction of test molecules with Fhm polypeptides may alsobe assayed directly using polyclonal or monoclonal antibodies in animmunoassay. Alternatively, modified forms of Fhm polypeptidescontaining epitope tags as described herein may be used in immunoassays.

[0333] Homogeneous assay technologies for radioactivity (SPA; Amersham)and time resolved fluorescence (HTRF, Packard) can also be implemented.Binding can be detected by labeling with radioactive isotopes (¹²⁵I,³⁵S, ³H), fluorescent dyes (fluorescein), lanthanides such as Europeum(Eu³⁻) chelates or cryptates, orbipyridyl-ruthenium (Ru²⁻) complexes. Itis understood that the choice of a labeled probe will depend upon thedetection system used. Alternatively, Fhm or putative Fhm agonists orantagonists may be modified with an unlabeled epitope tag (e.g., biotin,peptides, His6, myc, Fc) and bound to proteins such as streptavidin,anti-peptide or anti-protein antibodies which have a detectable label asdescribed above.

[0334] Binding of test molecules to putative Fhm receptor polypeptidesmay also be assayed directly using polyclonal or monoclonal antibodiesin an immunoassay. Alternatively, modified forms of putativeFhm-receptor polypeptide(s) containing epitope tags as described abovemay be used in solution and immunoassays.

[0335] In one embodiment, modulators of the Fhm-ligand may be a protein,peptide, carbohydrate, lipid or small molecular weight molecule.Potential protein antagonists of Fhm include antibodies which bind toactive regions of the polypeptide and inhibit or eliminate binding ofFhm to its putative receptor. Molecules which regulate Fhm polypeptideexpression may include nucleic acids which are complementary to nucleicacids encoding a Fhm polypeptide, or are complementary to nucleic acidssequences which direct or control expression of polypeptide, and whichact as anti-sense regulators of expression.

[0336] In the event that Fhm polypeptides display biological activitythrough an interaction with a binding partner (e.g., a receptor), avariety of in vitro assays may be used to measure binding of Fhmpolypeptide to a corresponding binding partner (such as a selectivebinding agent or ligand). These assays may be used to screen testmolecules for their ability to increase or decrease the rate and/or theextent of binding of a Fhm polypeptide to its binding partner. In oneassay, Fhm polypeptide is immobilized in the wells of a microtiterplate. Radiolabeled Fhm binding partner (for example, iodinated Fhmbinding partner) and the test molecule(s) can then be added either oneat a time (in either order) or simultaneously to the wells. Afterincubation, the wells can be washed and counted (using a scintillationcounter) for radioactivity to determine the extent of binding to whichthe binding partner bound to Fhm polypeptide. Typically, the moleculeswill be tested over a range of concentrations, and a series of controlwells lacking one or more elements of the test assays can be used foraccuracy in the evaluation of the results. An alternative to this methodinvolves reversing the “positions” of the proteins. i.e., immobilizingFhm binding partner to the microtiter plate wells, incubating with thetest molecule and radiolabeled Fhm and determining the extent of Fhmbinding (see, for example, Chapter 18 of Current Protocols in MolecularBiology, Ausubel et al., eds., John Wiley & Sons, New York, N.Y. ,1995).

[0337] As an alternative to radiolabeling, a Fhm polypeptide or itsbinding partner may be conjugated to biotin and the presence ofbiotinylated protein can then be detected using streptavidin linked toan enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase(AF), that can be detected colorometrically, or by fluorescent taggingof sireptavidin. An antibody directed to an Fhm polypeptide or to an Fhmbinding partner and is conjugated to biotin may also be used and can bedetected after incubation with enzyme-linked streptavidin linked to APor HRP

[0338] A Fhm polypeptide and a Fhm binding partner can also beimmobilized by attachment to agarose beads, acrylic beads or other typesof such inert solid phase substrates. The substrate-protein complex canbe placed in a solution containing the complementary protein and thetest compound. After incubation, the beads can be precipitated bycentrifugation, and the amount of binding between an Fhm polypeptide andits binding partner can be assessed using the methods described above.Alternatively, the substrate-protein complex can be immobilized in acolumn and the test molecule and complementary protein passed over thecolumn. Formation of a complex between an Fhm polypeptide and itsbinding partner can then be assessed using any of the techniquesdescribed herein, i.e., radiolabeling, antibody binding, or the like.

[0339] Another in vitro assay that is useful for identifying a testmolecule which increases or decreases the formation of a complex betweena Fhm binding protein and a Fhm binding partner is a surface plasmonresonance detector system such as the BIAcore assay system (Pharmacia,Piscataway, N.J.). The BIAcore system may be carried out using themanufacturer's protocol. This assay essentially involves the covalentbinding of either Fhm or a Fhm binding partner to a dextran-coatedsensor chip which is located in a detector. The test compound and theother complementary protein can then be injected either simultaneouslyor sequentially into the chamber containing the sensor chip. The amountof complementary proteinbinds can be assessed based on the change inmolecular mass which is physically associated with the dextran-coatedside of the sensor chip; the change in molecular mass can be measured bythe detector system.

[0340] In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease theformation of a complex between a Fhm polypeptide and a Fhm bindingpartner complex. In these cases, the assays described herein can bereadily modified by adding such additional test compound(s) eithersimultaneous with, or subsequent to, the first test compound. Theremainder of the steps in the assay are as set forth herein.

[0341] In vitro assays such as those described herein may be usedadvantageously to screen rapidly large numbers of compounds for effectson complex formation by Fhm and Fhm binding partner. The assays may beautomated to screen compounds generated in phage display, syntheticpeptide and chemical synthesis libraries.

[0342] Compounds which increase or decrease the formation of a complexbetween a Fhm polypeptide and a Fhm binding partner may also be screenedin cell culture using cells and cell lines expressing either Fhm or Fhmbinding partner. Cells and cell lines may be obtained from any mammal,but preferably will be from human or other primate, canine, or rodentsources. The binding of an Fhm polypeptide to cells expressing Fhmbinding partner at the surface is evaluated in the presence or absenceof test molecules and the extent of binding may be determined by, forexample, flow cytometry using a biotinylated antibody to an Fhm bindingpartner. Cell culture assays may be used advantageously to furtherevaluate compounds that score positive in protein binding assaysdescribed herein.

[0343] Cell cultures can also be used to screen the impact of a drugcandidate. For example, drug candidates may decrease or increase theexpression of the Fhm gene. In certain embodiments, the amount of Fhmpolypeptide that is produced may be measured after exposure of the cellculture to the drug candidate. In certain embodiments, one may detectthe actual impact of the drug candidate on the cell culture. Forexample, the overexpression of a particular gene may have a particularimpact on the cell culture. In such cases, one may test a drugcandidate's ability to increase or decrease the expression of the geneor its ability to prevent or inhibit a particular impact on the cellculture. In other examples, the production of a particular metabolicproduct such as a fragment of a polypeptide, may result in, or beassociated with, a disease or pathological condition. In such cases, onemay test a drug candidate's ability to decrease the production of such ametabolic product in a cell culture.

[0344] P38 Inhibitors

[0345] A new approach to intervention between the extracellular stimulusand the secretion of IL-1 and TNFα from the cell involves blockingsignal transduction through inhibition of a kinase which lies on thesignal pathway. One example is through inhibition of P-38 (also called“RK” or “SAPK-2”, Lee et al., Nature, 372:739, 1994), a known ser/thrkinase (clone reported in Han et al., Biochimica Biophisica Acta,1265:224-227, 1995). A linear relationship has been shown foreffectiveness in a competitive binding assay to P-38, and the sameinhibitor diminishing the levels of IL-1 secretion from monocytesfollowing LPS stimulation. Following LPS stimulation of monocytes, thelevels of messenger RNA for TNF-α have been shown to increase 100 fold,but the protein levels of TNF-α are increased 10,000 fold. Thus, aconsiderable amplification of the TNF signaling occurs at thetranslational level. Following LPS stimulation of monocytes in thepresence of a P-38 inhibitor, the levels of mRNA are not affected, butthe levels of final TNF protein are dramatically reduced (up to 80-90%depending on the effectiveness of the P-38 inhibitor). Thus, the aboveexperiments lend strong support to the conclusion that inhibition ofP-38 leads to diminished translational efficiency. Further evidence thatTNFa is under translational control is found in the deletion experimentsof Beutler et al. and Lee, wherein segments of 3′ untranslated mRNA (3′UTR) are removed resulting in high translational efficiency for TNFα.More importantly, the P-38 inhibitors did not have an effect on thelevel of TNFα (i.e., translational efficiency) when the appropriatesegments of TNFα mRNA are deleted. Thus, the correlative data betweenthe level of binding of inhibitors to P-38 and the diminished IL-1 andTNFα levels following LPS stimulation with the same inhibitors, plus theabove biochemical evidence regarding the effect of P-38 inhibitors ontranslational efficiency of both TNFα and IL-1 make a strong cause andeffect relationship. The role of P-38 in the cell is still beingdelineated; so therefore, other beneficial effects regardinginflammnatory diseases or other disease states obtained from itsinhibition maybe forthcoming.

[0346] Elevated levels of TNFα and/or IL-1 may contribute to the onset,etiology, or exacerbate a number of disease states, including, but notlimited to: rheumatoid arthritis; osteoarthritis; rheumatoidspondylitis; gouty arthritis; inflammatory bowel disease; adultrespiratory distress syndrome (ARDS); psoriasis; Crohn's disease;allergic rhinitis; ulcerative colitis; anaphylaxis; contact dermatitis;asthma; antiviral therapy including those viruses sensitive to TNFαinhibition—HIV-1, HIV-2, HIV-3, cytomegalovirus (CMV), influenza,adenovirus, and the herpes viruses including HSV-1, HSV-2, and herpeszoster; muscle degeneration; cachexia; Reiter's syndrome; type IIdiabetes; bone resorption diseases; graft vs. host reaction; ischemiareperfusion injury; atherosclerosis; brain trauma; Alzheimer's disease;multiple sclerosis; cerebral malaria; sepsis; septic shock; toxic shocksyndrome; fever and mylagias due to infection.

[0347] Substituted imidazole, pyrrole, pyridine, pyrimidine and the likecompounds have been described for use in the treatment of cytokinemediated diseases by inhibition of proinflammatory cytokines, such asIL-1, IL-6, IL-8 and TNF. Substituted imidazoles for use in thetreatment of cytokine mediated diseases have been described in U.S. Pat.No. 5,593,992; WO 93/14081; WO 97/18626; WO 96/21452; WO 96/21654; WO96/40143; WO 97/05878; WO 97/05878; (each of which is incorporatedherein by reference in its entirety). Substituted imidazoles for use inthe treatment of inflammation has been described in U.S. Pat. No.3,929,807 (which is incorporated herein by reference in its entirety).Substituted pyrrole compounds for use in the treatment of cytokinemediated diseases have been described in WO 97/05877; WO 97/05878; WO97/16426; WO 97/16441; and WO 97/16442 (each of which is incorporatedherein by reference in its entirety). Substituted aryl and heteroarylfused pyrrole compounds for use in the treatment of cytokine mediateddiseases have been described in WO 98/22457 (which is incorporatedherein by reference in its entirety). Substituted pyridine, pyrimidine,pyrimidinone and pyridazine compounds for use in the treatment ofcytokine mediated diseases have been described in WO 98/24780; WO98/24782; WO 99/24404; and WO 99/32448 (each of which is incorporatedherein by reference in its entirety).

[0348] Internalizing Proteins

[0349] The TAT protein sequence (from HIV) can be used to internalizeproteins into a cell by targeting the lipid bi-layer component of thecell membrane. See e.g., Falwell et al., Proc. Natl. Acad. Sci., 91:664-668, 1994. For example, an 11 amino acid sequence (YGRKKRRQRRR; SEQID NO: 22) of the HIV TAT protein (termed the “protein transductiondomain”, or TAT PDT) has been shown to mediate delivery of largebioactive proteins such as β-galactosidase and p27Kip across thecytoplasmic membrane and the nuclear membrane of a cell. See Schwarze etal., Science, 285: 1569-1572, 1999; and Nagahara et al., NatureMedicine, 4: 1449-1452, 1998. Schwartze et al. (Science, 285: 1569-72,1999) demonstrated that cultured cells acquired β-gal activity whenexposed to a fusion of the TAT PDT and β-galactosidase. Injection ofmice with the TAT-β-gal fusion proteins resulted in β-gal expression ina number of tissues, including liver, kidney, lung, heart, and braintissue.

[0350] It will thus be appreciated that the TAT protein sequence may beused to internalize a desired protein or polypeptide into a cell. In thecontext of the present invention, the TAT protein sequence can be fusedto another molecule such as a Fhm antagonist (i.e.: anti-Fhm selectivebinding agent or small molecule) and administered intracellularly toinhibit the activity of the Fhm molecule. Where desired, the Fhm proteinitself, or a peptide fragment or modified form of Fhm, may be fused tosuch a protein transducer for administrating to cells using theprocedures, described above.

[0351] Therapeutic Uses

[0352] Members of the TNF ligand family have been implicated inmediation of a number of diseases. The pleiotropic nature of TNF andrelated ligand family members prevents generalization about whether aparticular polypeptide is beneficial or injurious. It is clear that insome instances, the local effects of TNF and other members of theTNF-ligand ligand family of cytokines improve host defense mechanisms bymobilizing substrate, increasing immune cell function, stimulatinginflammation, and in killing cancer cells. However, in other cases thetoxicity of TNF and related cytokines may cause disease by mediatingshock, tissue injury, or catabolic injury. There are many diseaseswherein injury that is mediated by members of the TNF ligand family maybe treated or ameliorated by the administration of soluble forms ofmembers of the TNF-receptor gene family or TNF-like ligand molecules.These diseases include acquired-immunodeficiency syndrome (AIDS),anemia, autoimmune diseases, cachexia, cancer, cerebral malaria,diabetes mellitus, disseminated intravascular coagulopathy, erythryoidsick syndrome, hemorrhagic shock, hepatitis, insulin resistance,leprosy, leukemia, lymphoma, meningitis, multiple sclerosis, myocardialischaemia, obesity, rejection of transplanted organs, rheumatoidarthritis, septic shock syndrome, stroke, adult respiratory distresssyndrome (ARDS), tuberculosis, and a number of viral diseases.

[0353] Fhm Compositions and Administration

[0354] Pharmaceutical compositions of Fhm polypeptides are within thescope of the present invention for prophylactic and therapeutictreatment of humans and animals for indications resulting from abnormalexpression of Fhm or where it is determined that administration of Fhmpolypeptide will result in the amelioration or cure of the indications.Such compositions may comprise a therapeutically effective amount of aFhm polypeptide and/or its binding partner, or therapeutically activefragment(s), variant(s), or derivative(s) thereof in admixture with apharmaceutically acceptable additives and/or carriers. Suitableformulation materials or pharmaceutically acceptable agents include, butare not limited to, antioxidants, preservatives, colors, flavoring, anddiluting agents, emulsifying agents, suspending agents, solvents,fillers, bulking agents, buffers, delivery vehicles, diluents,excipients, and/or pharmaceutical adjuvants. Typically, a therapeuticcompound containing Fhm polypeptide(s) will be administered in the formof a composition comprising purified polypeptide, fragment(s),variant(s), or derivative(s) in conjunction with one or morephysiologically acceptable carriers, excipients, or diluents. Forexample, a suitable vehicle may be water for injection, physiologicalsolution, or artificial cerebrospinal fluid possibly supplemented withother materials common in compositions for parenteral delivery.

[0355] Neutral buffered saline or saline mixed with serum albumin areexemplary appropriate carriers. Preferably, the product is formulated asa lyophilizate using appropriate excipients (e.g., sucrose). Otherstandard carriers, diluents, and excipients may be included as desired.Other exemplary compositions comprise Tris buffer of about pH 7.0-8.5,or acetate buffer of about pH 4.0-5.5, which may further includesorbitol or a suitable substitute therefor. The pH of the solutionshould also be selected based on the relative solubility of Fhm atvarious pHs.

[0356] The primary solvent in a composition may be either aqueous ornon-aqueous in nature. In addition, the vehicle may contain otherformulation materials for modifying or maintaining the pH, osmolarity,viscosity, clarity, color, sterility, stability, isotonicity, rate ofdissolution, or odor of the formulation. Similarly, the composition maycontain additional formulation materials for modifying or maintainingthe rate of release of Fhm protein, or for promoting the absorption orpenetration of Fhm protein.

[0357] Compositions comprising the Fhm polypeptide compositions can beadministered parentally. Alternatively, the compositions may beadministered intravenously or subcutaneously. When systemicallyadministered, the therapeutic compositions for use in this invention maybe in the form of a pyrogen-free, parentally acceptable aqueoussolution. The preparation of such pharmaceutically acceptable proteinsolutions, with due regard to pH, isotonicity, stability and the like,is within the skill of the art.

[0358] Therapeutic formulations of Fhm polypeptide compositions usefulfor practicing the present invention may be prepared for storage bymixing the selected composition having the desired degree of purity withoptional physiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company, 1990) in the form of a lyophilized cake or anaqueous solution.

[0359] Acceptable carriers, excipients or stabilizers are nontoxic torecipients and are preferably inert at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, or otherorganic acids; antioxidants such as ascorbic acid; low molecular weightpolypeptides; proteins, such as serum, albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, pluronics orpolyethylene glycol (PEG).

[0360] An effective amount of the Fhm polypeptide(s) composition to beemployed therapeutically will depend, for example, upon the therapeuticobjectives such as the indication for which the composition is beingused, the route of administration (e.g., whether it is administeredlocally or systemically), and the condition of the patient (e.g.,patient's general health, anaureuesis, age, weight, sex). It isessential, when determining the therapeutically effective dose, to takeinto account the quantity of Fhm or other members of the TNF family thatare responsible for the disease. Basically, it can be assumed that foreffective treatment of a disease triggered by the over expression ofcytokine(s) such as Fhm, at least the same molar amount of the Fhmpolypeptide(s) is required, and possibly a multiple excess might beneeded, although less may be needed depending on the nature of thereceptor and the nature of its interaction with Fhm. Accordingly, itwill be necessary for the therapist to titer the dosage and/or in vivomodify the route of administration as required to obtain the optimaltherapeutic effect. A typical daily dosage may range from about 0.1mg/kg to up to 100 mg/kg or more, depending on the factors mentionedabove. Typically, a clinician will administer the composition until adosage is reached that achieves the desired effect. The composition maytherefore be administered as a single dose, or as two or more doses(which may or may not contain the same amount of Fhm polypeptide) overtime, or as a continuous infusion via implantation device or catheter.

[0361] As further studies are conducted, information will emergeregarding appropriate dosage levels for treatment of various conditionsin various patients, and the ordinary skilled worker, considering thetherapeutic context, the type of disorder under treatment, the age andgeneral health of the recipient, will be able to ascertain properdosing.

[0362] The Fhm polypeptide composition to be used for in vivoadministration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes. Where the compositionis lyophilized, sterilization using thes method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration ordinarily will be stored in lyophilizedform or in solution.

[0363] Therapeutic compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations may bestored either in a ready-to-use form or in a form (e.g., lyophilized)requiring reconstitution prior to administration.

[0364] Effective administration forms, such as (1) slow-releaseformulations, (2) inhalant mists, or (3) orally active formulations arealso envisioned. Pharmaceutical compositions comprising thereapeuticallyeffective dose of the Fhm polypeptide also may be formulated forparenteral administration. Such parenterally administered therapeuticcompositions are typically in the form of a pyrogen-free, parenterallyacceptable aqueous solution comprising Fhm in a pharmaceuticallyacceptable vehicle. The Fhm pharmaceutical compositions also may includeparticulate preparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc. or the introduction of Fhm into liposomes.Hyaluronic acid may also be used, and this may have the effect ofpromoting sustained duration in the circulation.

[0365] A particularly suitable vehicle for parenteral injection issterile distilled water in which Fhm is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation mayinvolve the formulation of Fhm with an agent, such as injectablemicrospheres, bio-erodible particles or beads, or liposomes, thatprovides for the controlled or sustained release of the protein productwhich may then be delivered as a depot injection. Other suitable meansfor the introduction of Fhm include implantable drug delivery deviceswhich contain the Fhm and/or its binding partner.

[0366] The preparations of the present invention may include othercomponents, for example parenterally acceptable preservatives, tonicityagents, cosolvents, wetting agents, complexing agents, buffering agents,antimicrobials, antioxidants and surfactants, as are well known in theart. For example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol and the like. Suitable preservatives include, but are notlimited to, benzalkonium chloride, thimerosal, phenethyl alcohol,methylparaben, propylparaben, chlorhexidine, sorbic acid and the like.Hydrogen peroxide may also be used as preservative. Suitable cosolventsare for example glycerin, propylene glycol and polyethylene glycol.Suitable complexing agents are for example caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal and the like. The bufferscan be conventional buffers such as borate, citrate, phosphate,bicarbonate, or Tris-HCl.

[0367] The formulation components are present in concentration that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pH,typically within a pH range of from about 5 to about 8.

[0368] When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired Fhm molecule in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which an Fhm molecule is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid or polyglycolic acid), or beads or liposomes,that provides for the controlled or sustained release of the productwhich may then be delivered via a depot injection. Hyaluronic acid mayalso be used, and this may have the effect of promoting sustainedduration in the circulation. Other suitable means for the introductionof the desired molecule include implantable drug delivery devices.

[0369] A pharmaceutical composition may be formulated for inhalation.For example, Fhm may be formulated as a dry powder for inhalation. Fhmpolypeptides or Fhm nucleic acid molecule inhalation solutions may alsobe formulated with a d propellant for aerosol delivery. In yet anotherembodiment, solutions may be nebulized. Pulmonary administration isfurther described in PCT Application No. PCT/US94/01875, which describespulmonary delivery of chemically modified proteins.

[0370] It is also contemplated that certain formulations containing Fhmpolypeptide(s) may be administered orally. In one embodiment, the Fhmligand which is administered in this fashion may be formulated with orwithout those carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. For example, a capsule may bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the Fhm polypeptide. Diluents, flavorings,low melting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be employed.

[0371] Another pharmaceutical composition may involve an effectivequantity of Fhm polypeptide in a mixture with non-toxic excipients whichare suitable for the manufacture of tablets. By dissolving the tabletsin sterile water, or other appropriate vehicle, solutions can beprepared in unit dose form. Suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

[0372] Additional Fhm-polypeptide pharmaceutical compositions will beevident to those skilled in the art, including formulations involvingFhm-polypeptide in sustained or controlled delivery formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, for example, PCT Application No.PCT/US93/00829 which describes the controlled release porous polymericmicroparticles for the delivery of pharmaceutical compositions.Additional examples include semipermeable polymer matrices in the formof shaped articles e.g., films or microspheres.

[0373] In a specific embodiment, the present invention is directed tokits for producing a single-dose administration unit. The kits may eachcontain both a first container having a dried protein and a secondcontainer having an aqueous formulation. Also included within the scopeof this invention are kits containing single and multi-chamberedpre-filled syringes (e.g., liquid syringes and lyosyringes).

[0374] The effective amount of an Fhm pharmaceutical composition to beemployed therapeutically will depend, for example, upon the therapeuticcontext and objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the Fhmmolecule is being used, the route of administration, and the size (bodyweight, body surface or organ size) and condition (the age and generalhealth) of the patient. Accordingly, the clinician may titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 mg/kg to up to about100 mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage may range from 0.1 mg/kg up to about 100 mg/kg;or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about 100 mg/kg.

[0375] The frequency of dosing will depend upon the pharmacokineticparameters of the Fhm molecule in the formulation used. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose, or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data.

[0376] The route of administration of the pharmaceutical composition isin accord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes, orroutes; by sustained releasesystems or by implantation devices. Where desired, the compositions maybe administered by bolus injection or continuously by infusion, or byimplantation device.

[0377] Alternatively or additionally, the composition may beadministered locally via implantation of a membrane, sponge, or anotherappropriate material on to which the desired molecule has been absorbedor encapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

[0378] One may further administer the present pharmaceuticalcompositions by pulmonary administration, see, e.g., InternationalPublication No: WO 94/20069, which discloses pulmonary delivery ofchemically modified proteins, herein incorporated by reference. Forpulmonary delivery, the particle size should be suitable for delivery tothe distal lung. For example, the particle size may be from 1 mm to 5mm, however, larger particles may be used, for example, if each particleis fairly porous. Alternatively or additionally, the composition may beadministered locally via implantation into the affected area of amembrane, sponge, or other appropriate material on to which receptorpolypeptide has been absorbed or encapsulated. Where an implantationdevice is used, the device may be implanted into any suitable tissue ororgan, and delivery may be directly through the device via bolus, or viacontinuous administration, or via catheter using continuous infusion.

[0379] Fhm-ligand polypeptide(s) and/or its binding partner may also beadministered in a sustained release formulation or preparation. Suitablepolymer compositions preferably have intrinsic and controllablebiodegradability so that they persist for about a week to about sixmonths; are non-toxic containing no significant toxic monomers anddegrading into non-toxic components; are biocompatible, are chemicallycompatible with substances to be delivered, and tend not to denature theactive substance; are sufficiently porous to allow the incorporation ofbiologically active molecules and their subsequent liberation from thepolymer by diffusion, erosion or a combination thereof; are able toremain at the site of the application by adherence or by geometricfactions, such as being formed in place or softened and subsequentlymolded or formed into microparticles , which are trapped at a desiredlocation; are capable of being delivered by techniques of minimuminvasivity such as by catheter, laparoscope or endoscope. Sustainedrelease matrices include polyesters, hydrogels, polylactides (U.S. Pat.No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al, Biopolymers, 22: 547-556, 1983), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15: 167-277, 1981 and Langer, Chem. Tech., 12: 98-105, 1982), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid(EP 133,988). Sustained-release compositions also may include liposomes,which can be prepared by any of several methods known in the art (e.g.,Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692. 1985; EP36,676; EP 88,046; EP 143,949, incorporated herein by reference).

[0380] The Fhm polypeptides, variants, derivatives or fragments thereof,may be employed alone, together, or in combination with otherpharmaceutical compositions. The Fhm polypeptides, fragments, variants,and derivatives may be used in combination with cytokines, cytokineinhibitors, growth factors, antibiotics, anti-inflammatories, and/orchemotherapeutic agents as is appropriate for the indication beingtreated In some cases, it may be desirable to use Fhm polypeptidepharmaceutical compositions in an ex vivo manner. In such instances,cells, tissues, or organs that have been removed from the patient areexposed to Fhm polypeptide pharmaceutical compositions after which thecells, tissues and/or organs are subsequently implanted back into thepatient.

[0381] In other cases, a Fhm polypeptide can be delivered by implantinginto patients certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptides, fragments, variants, or derivatives. Such cells may beanimal or human cells, and may autologous, heterologous or xenogeneic.Optionally, the cells may be immortalized. In order to decrease thechance of an immunological response, it is preferred that the cells maybe encapsulated to avoid infiltration of surrounding tissues. Theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

[0382] Methods used for membrane encapsulation of cells are familiar tothe skilled artisan, and preparation of encapsulated cells and theirimplantation in patients may be accomplished without undueexperimentation. See, e.g., U.S. Pat. Nos. 4,892,538; 5,011,472; and5,106,627, incorporated herein by reference. A system for encapsulatingliving cells is described in International Publication No: WO 91/10425.Techniques for formulating a variety of other sustained or controlleddelivery means, such as liposome carriers, bio-erodible particles orbeads, are also known to those in the art, and are described, forexample, in U.S. Pat. No.5,653,975, incorporated herein by reference.The cells, with or without encapsulation, may be implanted into suitablebody tissues or organs of the patient.

[0383] As discussed herein, it may be desirable to treat isolated cellpopulations such as stem cells, lymphocytes, red blood cells,chondrocytes, neurons, and the like; add as appropriate with one or moreFhm polypeptides, variants, derivatives and/or fragments. This can beaccomplished by exposing the isolated cells to the polypeptide, variant,derivative, or fragment directly, where it is in a form that ispermeable to the cell membrane.

[0384] The present invention relates to improved methods for both the invitro production of therapeutic proteins and for the production anddelivery of therapeutic proteins by gene therapy.

[0385] Homologous Recombination

[0386] It is further envisioned that Fhm protein may be produced byhomologous recombination, or with recombinant production methodsutilizing control elements introduced into cells already containing DNAencoding Fhm. For example, homologous recombination methods may be usedto modify a cell that contains a normally transcriptionally silent Fhmgene, or under expressed gene, and thereby produce a cell whichexpresses therapeutically efficacious amounts of Fhm. Homologousrecombination is a technique originally developed for targeting genes toinduce or correct mutations in transcriptionally active genes(Kucherlapati, Prog. in Nucl. Acid Res. and Mol. Biol., 36:301, 1989).The basic technique was developed as a method for introducing specificmutations into specific regions of the mammalian genome (Thomas et al.,Cell, 44:419-428, 1986; Thomas and Capecchi, Cell, 51:503-512, 1987;Doetschman et al., Proc. Natl. Acad. Sci., 85:8583-8587, 1988) or tocorrect specific mutations within defective genes (Doetschman et al.,Nature, 330:576-578, 1987). Exemplary homologous recombinationtechniques are described in U.S. Pat. No: 5,272,071, EP Publication No:91 90 3051, EP Publication No. 505 500; PCT/US90/07642, InternationalPublication No: WO 91/09955, incorporated herein by reference.

[0387] Through homologous recombination, the DNA sequence to be insertedinto the genome can be directed to a specific region of the gene ofinterest by attaching it to targeting DNA. The targeting DNA is anucleotide sequence that is complementary (homologous) to a region ofthe genomic DNA, into which insertion of the sequence is sought. Smallpieces of targeting DNA that are complementary to a specific region ofthe genome are put in contact with the parental strand during the DNAreplication process. It is a general property of DNA that has beeninserted into a cell to hybridize, and therefore, recombine with otherpieces of endogenous DNA through shared homologous regions. If thiscomplementary strand is attached to an oligonucleotide that contains amutation or a different sequence or an additional nucleotide, it too isincorporated into the newly synthesized strand as a result of therecombination. As a result of the proofreading function, it is possiblefor the new sequence of DNA to serve as the template. Thus, thetransferred DNA is incorporated into the genome.

[0388] Attached to these pieces of targeting DNA are regions of DNAwhich may interact with or control the expression of a Fhm polypeptide,e.g. flanking sequences. For example, a promoter/enhancer element, asuppresser, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredFhm polypeptide. The control element controls a portion of the DNApresent in the host cell genome. Thus, the expression of Fhm protein maybe achieved not by transfection of DNA that encodes the Fhm gene itself,but rather by the use of targeting DNA (containing regions of homologywith the endogenous gene of interest) coupled with DNA regulatorysegments that provide the endogenous gene sequence with recognizablesignals for transcription of a Fhm protein.

[0389] In an exemplary method, expression of a desired targeted gene ina cell (i.e., a desired endogenous cellular gene) is altered by theintroduction, by homologous recombination into the cellular genome at apreselected site, by the introduction of DNA which includes at least aregulatory sequence, an exon and a splice donor site. These componentsare introduced into the chromosomal (genomic) DNA in such a manner thatthis, in effect, results in the production of a new transcription unit(in which the regulatory sequence, the exon and the splice donor sitepresent in the DNA construct are operatively linked to the endogenousgene). As a result of the introduction of these components into thechromosomal DNA, the expression of the desired endogenous gene isaltered.

[0390] Altered gene expression, as described herein, encompassesactivating (or causing to be expressed) a gene which is normally silent(unexpressed) in the cell as obtained, as well as increasing theexpression of a gene which is not expressed at physiologicallysignificant levels in the cell as obtained. The embodiment s furtherencompass changing the pattern of regulation or induction such that itis different from the pattern of regualtion or induction that occurs inthe cell as obtained, and reducing (including eliminating) expression ofa gene which is expressed in the cell as obtained.

[0391] One method by which homologous recombination can be used toincrease, or cause, Fhm polypeptide production from a cell's endogenousFhm gene involves first using homologous recombination to place arecombination sequence from a site-specific recombination system (e.g.,Cre/loxP, FLP/FRT) (see, Sauer, Current Opinion In Biotechnology,5:521-527, 1994; and Sauer, Methods In Enzymology, 225:890-900, 1993)upstream (that is, 5′ to) of the cell's endogenous genomic Fhmpolypeptide coding region. A plasmid containing a recombination sitehomologous to the site that was placed just upstream of the genomic Fhmpolypeptide coding region is introduced into the modified cell linealong with the appropriate recombinase enzyme. This recombinase enzymecauses the plasmid to integrate, via the plasmid's recombination site,into the recombination site located just upstream of the genomic Fhmpolypeptide coding region in the cell line (Baubonis and Sauer, NucleicAcids Res., 21:2025-2029, 1993; and O'Gorman et al., Science,251:1351-1355, 1991). Any flanking sequences known to increasetranscription (e.g., enhancer/promoter, intron, or translationalenhancer), if properly positioned in this plasmid, would integrate insuch a manner as to create a new or modified transcriptional unitresulting in de novo or increased Fhm polypeptide production from thecell's endogenous Fhm gene.

[0392] A further method to use the cell line in which the site-specificrecombination sequence has been placed just upstream of the cell'sendogenous genomic Fhm polypeptide coding region is to use homologousrecombination to introduce a second recombination site elsewhere in thecell line's genome. The appropriate recombinase enzyme is thenintroduced into the two-recombination-site cell line, causing arecombination event (deletion, inversion, or translocation) (Sauer,Current Opinion In Biotechnology, supra, 1994; and Sauer, Methods InEnzymology, supra, 1993) that would create a new or modifiedtranscriptional unit resulting in de novo or increased Fhm polypeptideproduction from the cell's endogenous Fhm gene.

[0393] An additional approach for increasing, or causing, the expressionof Fhm polypeptide from a cell's endogenous Fhm gene involvesincreasing, or causing, the expression of a gene or genes (e.g.,transcription factors) and/or decreasing the expression of a gene orgenes (e.g., transcriptional repressors) in a manner which results in denovo or increased Fhm polypeptide production from the cell's endogenousFhm gene. This method includes the introduction of a non-naturallyoccurring polypeptide (e.g., a polypeptide comprising a sitespecificsite-specific DNA binding domain fused to a transcriptionalfactor domain) into the cell such that de novo or increased Fhmpolypeptide production from the cell's endogenous Fhm gene results.

[0394] The present invention further relates to DNA constructs useful inthe method of altering expression of a target gene. In certainembodiments, the exemplary DNA constructs comprise: (a) on or moretargeting sequence; (b) a regulatory sequence; (c) an exon; and (d) anunpaired splice-donor site. The targeting sequence in the DNA constructdirects the integration of elements (a)-(d) into a target gene in a cellsuch that the elements (b)-(d) are operatively linked to sequences ofthe endogenous target gene. In another embodiment, the DNA constructscomprise: (a) one or more targeting sequence, (b) a regulatory sequence,(c) an exon, (d) a splice-donor site, (e) an intron, and (f) asplice-acceptor site, wherein the targeting sequence directs theintegration of elements (a)-(f) such that the elements of (b)-(f) areoperatively linked to the endogenous gene. The targeting sequence ishomologous to the preselected site in the cellular chromosomal DNA withwhich homologous recombination is to occur. In the construct, the exonis generally 3′ of the regulatory sequence and the splice-donor site is3′ of the exon.

[0395] If the sequence of a particular gene is known, such as thenucleic acid sequence of Fhm presented herein, a piece of DNA that iscomplementary to a selected region of the gene can be synthesized orotherwise obtained, such as by appropriate restriction of the native DNAat specific recognition sites bounding the region of interest. Thispiece serves as a targeting sequence(s) upon insertion into the cell andwill hybridize to its homologous region within the genome. If thishybridization occurs during DNA replication, this piece of DNA, and anyadditional sequence attached thereto, will act as an Okazaki fragmentand will be incorporated into the newly synthesized daughter strand ofDNA. The present invention, therefore, includes nucleotides encodingFhm-polypeptide(s), which nucleotides may be used as targetingsequences.

[0396] Alternatively, gene therapy can be employed as described below.

[0397] Fhm Cell Therapy and Gene Therapy

[0398] Fhm cell therapy, e.g., the implantation of cells producing Fhm,is also encompassed by the present invention. This embodiment involvesimplanting cells capable of synthesizing and secreting a biologicallyactive form of the soluble Fhm. Such soluble Fhm polypeptide producingcells may be cells that are natural producers of Fhm or may berecombinant cells whose ability to produce Fhm has been augmented bytransformation with a gene encoding the desired Fhm molecule or with agene augmenting the expression of Fhm polypeptide. Such a modificationmay be accomplished by means of a vector suitable for delivering thegene as well as promoting its expression and secretion. In order tominimize a potential immunological reaction in patients beingadministered a Fhm polypeptide as may occur with the adminstration of apolypeptide of a foreign species, it is preferred that the natural cellsproducing Fhm be of human origin and produce human Fhm. Likewise, it ispreferred that the recombinant cells producing Fhm be transformed withan expression vector containing a gene encoding a human Fhm polypeptide.

[0399] Implanted cells may be encapsulated to avoid the infiltration ofsurrounding tissue. Human or non-human animal cells may be implanted inpatients in biocompatible, semipermeable polymeric enclosures ormembranes that allow release of Fhm polypeptide but that prevent thedestruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissue. Alternatively, thepatient's own cells, transformed to produce Fhm polypeptides ex vivo,may be implanted directly into the patient without such encapsulation.

[0400] Techniques for the encapsulation of living cells are known in theart, and the preparation of the encapsulated cells and theirimplantation in patients may be accomplished without undueexperimentation. For example, Baetge et al. (WO 95105452 andPCT/US94/09299) describe membrane capsules containing geneticallyengineered cells for the effective delivery of biologically activemolecules. The capsules are biocompatible and are easily retrievable.The capsules are biocompatible and are easily retrievable. The capsulesencapsulate cells transfected with recombinant DNA molecules comprisingDNA sequences coding for biologically active molecules operativelylinked to promoters that are not subject to down-regulation in vivo uponimplantation into a mammalian host. The devices provide for the deliveryof the molecules from living cells to specific sites within a recipient.In addition, see U.S. Pat. Nos. 4,892,538, 5,011,472, and 5,106,627,incorporated herein by reference. A system for encapsulating livingcells is described in International Application WO 91/10425 of Aebischeret al., International Application No. WO 91/10470 of Aebischer et al.;Winn et al., Exper. Neurol., 113:322-329, 1991, Aebischer et al., Exper.Neurol., 11 1:269-275, 1991; and Tresco et al., ASAIO, 38:17-23, 1992,incorporated herein by reference.

[0401] In vivo and in vitro gene therapy delivery of Fhm is alsoencompassed by the present invention. In vivo gene therapy may beaccomplished by introducing the gene encoding Fhm into cells via localinjection of a polynpcleotide molecule or other appropriate deliveryvectors. (Hefti, J. Neurobiology, 25:1418-1435, 1994). For example, apolynucleotide molecule encoding Fhm may be contained in anadeno-associated virus vector for delivery to the targeted cells (Seefor e.g., International Publication No. WO 95/34670; InternationalApplication No. PCT/US95/07178). The recombinant adeno-associated virus(AAV) genome typically contains AAV inverted terminal repeats flanking aDNA sequence encoding Fhm operably linked to functional promoter andpolyadenylation sequences.

[0402] Alternative viral vectors include, but are not limited to,retrovirus, adenovirus, herpes simplex virus and papilloma virusvectors. U.S. Pat. No. 5.672,344 (issued Sep. 30, 1997, Kelley et al.,University of Michigan) describes an in vivo viral-mediated genetransfer system involving a recombinant neurotrophic HSV-1 vector. U.S.Pat. No. 5,399,346 (issued Mar. 21, 1995, Anderson et al., Department ofHealth and human Services) provides examples of a process for providinga patient with a therapeutic protein by the delivery of human cellswhich have been treated in vitro to insert a DNA segment encoding atherapeutic protein. Additional methods and materials for the practiceof gene therapy techniques are described in U.S. Pat. No. 5,631,236(issued May 20, 1997, Woo et al., Baylor College of Medicine) involvingadenoviral vectors; U.S. Pat. No. 5,672,510 (issued Sep. 30, 1997,Eglitis et al., Genetic Therapy, Inc.) involving retroviral vectors; andU.S. Pat. No. 5,635,399 (issued Jun. 3, 1997, Kriegler et al., ChironCorporation) involving retroviral vectors expressing cytokines.

[0403] Nonviral delivery methods include liposome-mediated transfer,naked DNA delivery (direct injection), receptor-mediated transfer(ligand-DNA complex), electroporation, calcium phosphate precipitationand microparticle bombardment (e.g., gene gun). Gene therapy materialsand methods may also include inducible promoters, tissue-specificenhancer-promoters, DNA sequences designed for site-specificintegration, DNA sequences capable of providing a selective advantageover the parent cell, labels to identify transformed cells, negativeselection systems and expression control systems (safety measures),cell-specific binding agents (for cell targeting), cell-specificinternalization factors, transcription factors to enhance expression bya vector as well as methods of vector manufacture. Such additionalmethods and materials for the practice of gene therapy techniques aredescribed in U.S. Pat. No. 4,970,154 (issued Nov. 13, 1990, D. C. Chang,Baylor College of Medicine) electroporation techniques; InternationalApplication No. WO 9640958 (published 961219, Smith et al., BaylorCollege of Medicine) nuclear ligands; U.S. Pat. No. 5,679,559 (issuedOct. 21, 1997, Kim et al., University of Utah Research Foundation)concerning a lipoprotein-containing system for gene delivery; U.S. Pat.No. 5,676,954 (issued Oct. 14, 1997, K. L. Brigham, VanderbiltUniversity involving liposome carriers; U.S. Pat. No. 5,593,875 (issuedJan. 14, 1997, Wurm et al., Genentech, Inc.) concerning methods forcalcium phosphate transfection; and U.S. Pat. No. 4,945,050 (issued Jul.31, 1990, Sanford et al., Cornell Research Foundation) whereinbiologically active particles are propelled at cells at a speed wherebythe particles penetrate the surface of the cells and become incorporatedinto the interior of the cells. Expression control techniques includechemical induced regulation (e.g., International Application Nos. WO9641865 and WO 9731899), the use of a progesterone antagonist in amodified steroid hormone receptor system (e.g., U.S. Pat. No.5,364,791), ecdysone control systems (e.g., International ApplicationNo. WO 9637609), and positive tetracycline-controllable transactivators(e.g., U.S. Pat. Nos. 5,589,362; 5,650,298; and 5,654,168).

[0404] It is also contemplated that Fhm gene therapy or cell therapy canfurther include the delivery of a second protein. For example, the hostcell may be modified to express and release soluble forms of both Fhmand TNF-α, or Fhm and IL-1. Alternatively, the Fhm and TNF-α, or Fhm andIL-1, may be expressed in and released from separate cells. Such cellsmay be separately introduced into the patient or the cells may becontained in a single implantable device, such as the encapsulatingmembrane described above.

[0405] One manner in which gene therapy can be applied is to use the Fhmgene (either genomic DNA, cDNA, and/or synthetic DNA encoding a Fhmpolypeptide, or a fragment, variant, or derivative thereof) which may beoperably linked to a constitutive or inducible promoter to form a “genetherapy DNA construct”. The promoter may be homologous or heterologousto the endogenous Fhm gene, provided that it is active in the cell ortissue type into which the construct will be inserted. Other componentsof the gene therapy DNA construct may optionally include, as required,DNA molecules designed for site-specific integration (e.g., endogenousflanking sequences useful for homologous recombination), tissue-specificpromoter, enhancer(s) or silencer(s), DNA molecules capable of providinga selective advantage over the parent cell, DNA molecules useful aslabels to identify transformed cells, negative selection systems, cellspecific binding agents (for example, for cell targeting) cell-specificinternalization factors, and transcription factors to enhance expressionby a vector as well as factors to enable vector manufacture.

[0406] A gene therapy DNA construct can then be introduced into thepatient's cells (either ex vivo or in vivo) using viral or non-viralvectors. One means for introducing the gene therapy DNA construct.Certain vectors, such as retroviral vectors, will deliver the DNAconstruct to the chromosomal DNA of the cells, and the gene canintegrate into the chromosomal DNA. Other vectors will function asepisomes, and the gene therapy DNA construct will remain in thecytoplasm. The use of gene therapy vectors is described, for example, inU.S. Pat. Nos. 5,672,344; 5,399,346; 5,631,236; and 5,635,399,incorporated herein by reference.

[0407] In yet other embodiments, regulatory elements can be included forthe controlled expression of the Fhm gene in the target cell. Suchelements are turned on in response to an appropriate effector. In thisway, a therapeutic polypeptide can be expressed when desired. Oneconventional control means involves the use of small molecule dimerizersor rapalogs (as described in WO 9641865 (PCT/US96/099486); WO 9731898(PCT/US97/03137) and WO9731899 (PCT/US95/03157)WO9731899(PCT/US95/03157)) used to dimerize chimeric proteins which contain asmall molecule-binding domain and a domain capable of initiatingbiological process, such as a DNA-binding protein or a transcriptionalactivation protein. The dimerization of the proteins can be used toinitiate transcription of the transgene.

[0408] An alternative regulation technology uses a method of storingproteins expressed from the gene of interest inside the cell as anaggregate or cluster. The gene of interest is expressed as a fusionprotein that includes a conditional aggregation domain which results inthe retention of the aggregated protein in the endoplasmic reticulum.The stored proteins are stable and inactive inside the cell. Theproteins can be released, however, by administering a drug (e.g., smallmolecule ligand) that removes the conditional aggregation domain andthereby specifically breaks apart the aggregates or clusters so that theproteins may be secreted from the cell. See, Science 287:816-817, and826-830 (2000).

[0409] Other suitable control means or gene switches include, but arenot limited to, the following systems. Mifepristone (RU486) is used as aprogesterone antagonist. The binding of a modified progesterone receptorligand-binding domain to the progesterone antagonist activatestranscription by forming a dimer of two transcription factors which thenpass into the nucleus to bind DNA. The ligand bindingligand-bindingdomain is modified to eliminate the ability of the receptor to bind tothe natural ligand. The modified steroid hormone receptor system isfurther described in U.S. Pat. No. 5,364,791; WO9640911, and WO9710337,WO 9640911 and WO 9710337.

[0410] Yet another control system uses ecdysone (a fruit fly steroidhormone) which binds to and activates an ecdysone receptor (cytoplasmicreceptor). The receptor then translocates to the nucleus to bind aspecific DNA response element (promoter from ecdysone-responsive gene).The ecdysone receptor includes a transactivation domain/DNA-bindingdomain/ligand-binding domain to initiate transcription. The ecdysonesystem is further described in U.S. Pat. No. 5,514,578; WO9738117;WO9637609;WO 9738117; WO 9637609 and WO9303162.

[0411] Another control means uses a positive tetracycline-controllabletransactivator. This system involves a mutated tet repressor proteinDNA-binding domain (mutated tet R-4 amino acid changes which resulted ina reverse tetracycline-regulated transactivator protein, i.e., it bindsto a tet operator in the presence of tetracycline) linked to apolypeptide which activates transcription. Such systems are described inU.S. Pat. Nos. 5,464,758; 5,650,298 and 5,654,168.

[0412] Additional expression control systems and nucleic acid constructsare described in U.S. Pat. Nos. 5,741,679 and 5,834,186, to InnovirLaboratories Inc.

[0413] In vivo gene therapy may be accomplished by introducing the geneencoding an Fhm polypeptide into cells via local injection of an Fhmnucleic acid molecule or by other appropriate viral or non-non-viraldelivery vectors. (Hefti, Neurobiology, 25:1418-1435, 1994). Forexample, a nucleic acid molecule encoding an Fhm polypeptide may becontained in an adeno-associated virus (AAV) vector for delivery to thetargeted cells (e.g., Johnson, International Publication No. WO95/34670;and International Application No. PCT/US95/07178). The recombinant AAVgenome typically contains AAV inverted terminal repeats flanking a DNAsequence encoding an Fhm polypeptide operably linked to functionalpromoter and polyadenylation sequences.

[0414] Alternative suitable viral vectors include, but are not limitedto, retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitisvirus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus,rhabdovirus, paramyxovirus, and papilloma virus vectors. U.S. Pat. No.5,672,344 describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5.399,346 provides examples of a process for providing a patient with atherapeutic protein by the delivery of human cells which have beentreated in vitro to insert a DNA segment encoding a therapeutic protein.Additional methods and materials for the practice of gene therapytechniques are described in U.S. Pat. No. 5,631.236 involving adenoviralvectors; U.S. Pat. No. 5,672,510 involving retroviral vectors; and U.S.Pat. No. 5,635,399 involving retroviral vectors expressing cytokines.

[0415] Nonviral delivery methods include, but are not limited to,liposome-mediated transfer, naked DNA delivery (direct injection),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,gene gun). Gene therapy materials and methods may also include the useof inducible promoters, tissue-specific enhancer-promoters, DNAsequences designed for site-specific integration, DNA sequences capableof providing a selective advantage over the parent cell, labels toidentify transformed cells, negative selection systems and expressioncontrol systems (safety measures), cell-specific binding agents (forcell targeting), cell-specific internalization factors, andtranscription factors to enhance expression by a vector as well asmethods of vector manufacture. Such additional methods and materials forthe practice of gene therapy techniques are described in U.S. Pat. No.4,970,154 involving electroporation techniques; WO96/40958 involvingnuclear ligands; U.S. Pat. No. 5,679,559 describing alipoprotein-containing system for gene delivery; U.S. Pat. No. 5,676,954involving liposome carriers; U.S. Pat. No. 5,593,875 concerning methodsfor calcium phosphate transfection; and U.S. Pat. No. 4,945,050 whereinbiologically active particles are propelled at cells at a speed wherebythe particles penetrate the surface of the cells and become incorporatedinto the interior of the cells.

[0416] A means to increase endogenous Fhm polypeptide expression in acell via gene therapy is to insert one or more enhancer elements intothe Fhm polypeptide promoter, where the enhancer element(s) can serve toincrease transcriptional activity of the Fhm polypeptides gene. Theenhancer element(s) used will be selected based on the tissue in whichone desires to activate the gene(s); enhancer elements known to conferpromoter activation in that tissue will be selected. For example, if aFhm gene encoding a Fhm polypeptide is to be “turned on” in T-cells, thelck promoter enhancer element may be used. Here, the functional portionof the transcriptional element to be added may be inserted into afragment of DNA containing the Fhm polypeptide promoter (and optionally,inserted into a vector, 5′ and/or 3′ flanking sequence(s), etc.) usingstandard cloning techniques. This construct, known as a “homologousrecombination construct”, can then be introduced into the desired cellseither ex vivo or in vivo.

[0417] Gene therapy also can be used to decrease Fhm polypeptideexpression where desired by modifying the nucleotide sequence of theendogenous promoter(s). Such modification is typically accomplished viahomologous recombination methods. For example, a DNA molecule containingall or a portion of the promoter of the Fhm gene(s) selected forinactivation can be engineered to remove and/or replace pieces of thepromoter that regulate transcription. For example, the TATA box and/orthe binding site of a transcriptional activator of the promoter may bedeleted using standard molecular biology techniques; such deletion caninhibit promoter activity thereby repressing the transcription of thecorresponding Fhm gene. The deletion of the TATA box or thetranscription activator binding site in the promoter may be accomplishedby generating a DNA construct comprising all or the relevant portion ofthe Fhm polypeptide promoter(s) (from the same or a related species asthe Fhm gene(s) to be regulated) in which one or more of the TATA boxand/or transcriptional activator binding site nucleotides are mutatedvia substitution, deletion and/or insertion of one or more nucleotides.As a result, the TATA box and/or activator binding site has decreasedactivity or is rendered completely inactive. The construct, which alsowill typically contain at least about 500 bases of DNA that correspondto the native (endogenous) 5′ and 3′ DNA sequences adjacent to thepromoter segment that has been modified. The construct may be introducedinto the appropriate cells (either ex vivo or i)z vivo) either directlyor via a viral vector as described herein. Typically, the integration ofthe construct into the genomic DNA of the cells will be via homologousrecombination, where the 5′ and 3′ DNA sequences in the promoterconstruct can serve to help integrate the modified promoter region viahybridization to the endogenous chromosomal DNA.

[0418] Addititional Uses of Fhm Nucleic Acids and Polypeptides

[0419] Nucleic acid molecules of the present invention (including thosethat do not themselves encode biologically active polypeptides) may beused to map the locations of the Fhm gene and related genes onchromosomes. Mapping may be done by techniques known in the art, such asPCR amplification and in situ hybridization.

[0420] Fhm nucleic acid molecules (including those that do notthemselves encode biologically active polypeptides),.may be useful ashybridization probes in diagnostic assays to test, either qualitativelyor quantitatively, for the presence of an Fhm DNA or corresponding RNAin mammalian tissue or bodily fluid samples.

[0421] The Fhm polypeptides may be used (simultaneously or sequentially)in combination with one or more cytokines, growth factors, antibiotics,anti-inflammatories, and/or chemotherapeutic agents as is appropriatefor the indication being treated.

[0422] Other methods may also be employed where it is desirable toinhibit the activity of one or more Fhm polypeptides. Such inhibitionmay be effected by nucleic acid molecules which are complementary to andwhich hybridize to expression control sequences (triple helix formation)or to Fhm mRNA. For example, antisense DNA or RNA molecules, which havea sequence that is complementary to at least a portion of the selectedFhm gene(s) can be introduced into the cell. Anti-sense probes may bedesigned by available techniques using the sequence of Fhm polypeptidedisclosed herein. Typically, each such antisense molecule will becomplementary to the start site (5′ end) of each selected Fhm gene. Whenthe antisense molecule then hybridizes to the corresponding Fhm mRNA,translation of this mRNA is prevented or reduced. Anti-sense inhibitorsprovide information relating to the decrease or absence of an Fhmpolypeptide in a cell or organism.

[0423] Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more Fhm polypeptides. In thissituation, the DNA encoding a mutant polypeptide of each selected Fhmpolypeptide can be prepared and introduced into the cells of a patientusing, either viral or non-viral methods as described herein. Each suchmutant is typically designed to compete with endogenous polypeptide inits biological role.

[0424] In addition, an Fhm polypeptide, whether biologically active ornot, may be used as an immunogen, that is, the polypeptide contains atleast one epitope to which antibodies may be raised. Selective bindingagents that bind to an Fhm polypeptide (as described herein) may be usedfor in vivo and in vitro diagnostic purposes, including, but not limitedto, use in-labeled form to detect the presence of Fhm polypeptide in abody fluid or cell sample. The antibodies may also be used to prevent,treat, or diagnose a number of diseases and disorders, including thoserecited herein. The antibodies may bind to an Fhm polypeptide so as todiminish or block at least one activity characteristic of an Fhmpolypeptide, or may bind to a polypeptide to increase at least oneactivity characteristic of an Fhm polypeptide (including by increasingthe pharmacokinetics of the Fhm polypeptide).

[0425] cDNA encoding Fhm polypeptide in E. coli was deposited with theATCC on ______ and having ATCC accession no. ______.

[0426] The following examples are intended for illustration purposesonly, and should not be construed as limiting the scope of the inventionin any way

EXAMPLE1 Isolation of DNA Encoding Human Fhm

[0427] A TNF family profile search of the Amgen expressed sequence tag(EST) database identified an EST clone designated Fhm1-00016-g12 from ahuman macrophage cDNA library encoding a potential TNF ligand familymember. A full-length cDNA encoding Fhm was obtained by PCR of firststrand cDNA prepared from the 5637 cell line (ATCC # HTB-9) using thefollowing primers:

[0428] 1406-53: 5′ GCCGAGGATCTGGGA CTGA (SEQ ID NO:1)

[0429] 1468-66 5′ TCGCCAATCCTCCAACCCATCTTA (SEQ ID NO:2)

[0430] The Fhm cDNA comprises 819 nucleotides (SEQ ID NO: 3) and encodesa polypeptide comprising 251 amino acids (SEQ ID NO: 4). Fasta search ofthe SwissProt database with the predicted Fhm protein sequence indicatedthat it is mostly related to TNFa with 28% identity in the C-terminal162 amino acid overlap. Like other TNF ligand family members. Fhm is atype II transmembrane protein, containing a short N-terminalintracellular domain (amino acids 1-36), a hydrophobic transmembraneregion (amino acids 37-56) and a long C-terminal extracellular domain(amino acids 57-251). The C-terminal extracellular domain of Fhmcontained most of the conserved region of the TNF ligand family (Smithet al., Cell 76:952-62, 1994).

EXAMPLE 2 Tissue Specific Expression of Fhm

[0431] Tissue specific expression patterns of Fhm gene may beinvestigated by Northern blot analysis using a ³²P-labeled PCR productas a probe to detect the presence of Fhm transcript in various tissues.

[0432] Cytoplasmic and poly-A+RNA is isolated from placenta, developingembryos, and various adult tissues using standard techniques Sambrook,J. et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, NewYork (1989). Cells/tissues are lysed with 20 ml of TRIzol reagent (BRL),homogenized for 30 seconds, and extracted with 4 ml of chloroform. Thetubes were centrifuged at 4000 rpm for 30 minutes and the aqueous phasewas transferred to new tubes. RNA was precipitated by adding 10 mlisopropanol, mixing, and centrifuging for 30 minutes at 4200 rpm. TheRNA pellet was washed with 10 ml of 70% ethanol, dried briefly, andresuspended in 0.5 ml TE buffer. Poly A⁺ RNA is prepared by using acommercially available mRNA purification kit (Pharmacia). After elutionof poly A⁻ RNA from the column in 750 μl of TE buffer, the sample wasthen ethanol precipitated by adding 40 μl sample buffer and 1 ml ethanoland maintaining at −70° C. overnight. Poly A⁺ RNA was then fractionatedusing a formaldehyde/agarose gel electrophoresis system. Followingelectrophoresis, the gel is processed and the RNA transferred to a nylonmembrane. See Sambrook et al. Supra. Northern blots are thenprehybridized in 20 ml of prehybridization solution containing 5×SSPE,50% formamide, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml denaturedsalmon sperm DNA for 2-4 hours at 42° C. The blots were then hybridizedin 20 ml of hybridization solution containing 6×SSPE, 50% formamide, 5×Denhardt's solution, 0.5% SDS, 100 ug/ml denatured salmon sperm DNA.Approximately 5 ng/ml of random primed, ³²P-labeled (RadPrime Kit,GEBCO) Fhm full length cDNA was used as a probe. The blots werehybridized for 18-24 hours at 42° C. The blots were then washed in0.1×SSC. 0.1% SDS at 55° C. The blots were then exposed to x-ray filmsfor three days at 80° C. A weak expression of Fhm was detected in thekidneys.

EXAMPLE 3 Production of Fhm Polypeptides

[0433] A. Expression of Fhm Polypeptide in Bacteria

[0434] PCR are used to amplify template DNA sequences encoding an Fhmpolypeptide using primers corresponding to the 5′ and 3′ ends of thesequence. The amplified DNA products may be modified to containrestriction enzyme sites to allow for insertion into expression vectors.PCR products are gel purified and inserted into expression vectors usingstandard recombinant DNA methodology. An exemplary vector, such aspAMG21 (ATCC No. 98113) containing the lux promoter and a gene encodingkanamycin resistance is digested with BamHI and NdeI for directionalcloning of inserted DNA. The ligated mixture is transformed into E. colihost strain 393 by electroporation and transformants selected forkanamycin resistance. Plasmid DNA from selected colonies is isolated andsubjected to DNA sequencing to confirm the presence of the insert.

[0435] Transformed host cells are incubated in 2XYT medium containing 30μg/ml kanamycin at 30° C. prior to induction. Gene expression can thenbe induced by addition of N-(3-oxohexanoyl)-dl-homoserine lactone to afinal concentration of 30 ng/ml followed by incubation at either 30° C.or 37° C. for six hours. Expression of Fhm polypeptide is evaluated bycentrifugation of the culture, resuspension and lysis of the bacterialpellets, and analysis of host cell proteins by SDS-polyacrylamide gelelectrophoresis.

[0436] Inclusion bodies containing Fhm polypeptide are purified asfollows: Bacterial cells are pelleted by centrifugation and resuspendedin water. The cell suspension is lysed by sonication and pelleted bycentrifugation at 195,000×g for 5 to 10 minutes. The supernatant isdiscarded and the pellet washed and transferred to a homogenizer. Thepellet is homogenized in 5 ml. of a Percoll solution (75% liquidPercoll. 0.15M NaCl) until uniformly suspended and then diluted andcentrifuged at 21,600×g for 30 minutes. Gradient fractions containingthe inclusion bodies are recovered and pooled. The isolated inclusionbodies are analyzed by SDS-PAGE. Recombinant Fhm protein was purified aspreviously described (WO 98/4675 1)

EXAMPLE 4 Production of Anti-Fhm Antibodies

[0437] Antibodies to Fhm polypeptides may be obtained by immunizationwith purified Fhm protein or with Fhm peptides produced by biological orchemical synthesis. Substantially pure Fhm protein or polypeptide may beisolated from transfected cells as described in Example 3. Concentrationof protein in the final preparation may be adjusted, for example, byconcentration on an amicon filter device, to the level of a fewmicrograms/ml. Monoclonal or polyclonal antibodies to the protein canthen be prepared by any of the procedures known in the art forgenerating antibodies such as those described in Hudson and Bay,“Practical Immunology, Second Edition”, Blackwell ScientificPublications, incorporated herein by reference.

[0438] Polyclonal antiserum containing antibodies to heterogenousepitopes of a single protein can be prepared by immunizing suitableanimals with the expressed protein described above, which can beunmodified or modified to enhance immunogenicity. Effective polyclonalantibody production is affected by many factors related both to theantigen and the host species. For example, small molecules tend to beless immunogenic than large molecules and may require the use ofcarriers or adjuvants. Also, host animals vary in response to site ofinoculations and dose, with both inadequate or excessive doses ofantigen resulting in low titer antisera. Small doses (ng levels) ofantigen administered at multiple intradermal sites appear to be mostreliable. An effective immunization protocol for rabbits can be found inVaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33: 988-991, 1971.

[0439] Booster injections can be given at regular intervals, andantiserum harvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begin to fall. See, forexample, Ouchterlony, O. et al., Chap. 19 in: Handbook of ExperimentalImmunology ed. D. Weir, Blackwell, 1973. Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12um). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chapt. 42 in; Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,eds.) Amer. Soc. For Microbiol., Washington, D.C., 1980.

[0440] Alternative procedures for obtaining anti-Fhm antibodies may alsobe employed, such as immunization of transgenic mice harboring human Igloci for production of fully human antibodies, and screening ofsynthetic antibody libraries, such as those generated by mutagenesis ofan antibody variable domain.

EXAMPLE 5 Functional Analysis of the Role of Fhm

[0441] To determine the functional role of Fhm in vivo, the Fhm gene iseither over expressed in the germ line of animals or inactivated in thegerm line of mammals by homologous recombination. See, .e.g, U.S. Pat.No. 5,489,743 and Interantional Patent Publication No. WO 94/28122,incorporated herein by reference. Animals in which the gene is overexpressed under the regulatory control of exogenous or endogenouspromoter elements are known as transgenic animals. Animals in which anendogenous gene has been inactivated by homologous recombination arealso known as “knockout” animals. Exemplary mammals include rabbits androdent species such as mice.

[0442] Transgenic animals allow for the determination of the effect(s)of over expression or inappropriate expression of the Fhm on developmentand disease processes. Fhm transgenic animals can also serve as a modelsystem to test compounds that can modulate receptor mediated Fhmactivity.

[0443] The “knockout” animals allow for the determination of the role ofFhm in embryonic development, and in immune and proliferative responses.The role of Fhm in development, and in immune and proliferative responseis determined by analysis the effect(s) of gene knockout on thedevelopment of the embryo as well as on the development anddifferentiation of various organs and tissues such as the immune systemin these animals. (as determined by FACS analysis of cell populations atdifferent stages of development).

EXAMPLE 6 Specific Recognition of Fhm by Soluble TNF-receptor FamilyMember NTR3

[0444] For receptor binding assay, 2×10⁵ COS-7 cells were seeded in6-well plate. The next day, cells were transfected with expressionplasmid for Fhm by lipofectamin methods according to the manufacturer'sinstructions (Gibco BRL). The eukaryotic expression vector PCEP4(Invitrogen) was used to generate the cDNA contstruct. After 48 hours oftransfection, the tissue culture medium was replaced with tissue culturemedium containing TNF-receptor family member(s) fused with human IgG Fcportion. After I hour incubation at room temperature (RT), cells werewashed three times with 5 ml PBS. Cells were then incubated in DMEMmedium containing 5% BSA and 1:500 dilution of goat anti-human IgG Fcconjugated with alkaline phosphatase (Sigma) for another hour at RT.After three washes with 5 ml TBS buffer, cells were stained with FastRed TR/AS-MX Substrate Kit (Pierce). Positive staining was determined byvisual examination under microscope. Fhm transfected COS-7 cells werespecifically recognized by NTR3Fc fusion protein. The NTR3 protein, amember of the TNF receptor supergene family, is described in detail inco-owned, co-filed provisional U.S. patent application filed Aug. 4,1999, Attorney Docket No. 01017/35549, incorporated herein by referencein its entirety.

[0445] While the present invention has been described in terms of thepreferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationswhich come within the scope of the invention as claimed.

1 22 1 19 DNA Artificial Sequence Description of Artificial Sequence PCRPrimer for human FHM 1 gccgaggatc tgggactga 19 2 24 DNA ArtificialSequence Description of Artificial Sequence PCR Primer for human FHM 2tcgccaatcc tccaacccat ctta 24 3 819 DNA Homo sapiens CDS (37)..(789)Hu-Fhm 3 aagctgggta cagctgctag caagctctag accacc atg gcc gag gat ctg gga54 Met Ala Glu Asp Leu Gly 1 5 ctg agc ttt ggg gaa aca gcc agt gtg gaaatg ctg cca gag cac ggc 102 Leu Ser Phe Gly Glu Thr Ala Ser Val Glu MetLeu Pro Glu His Gly 10 15 20 agc tgc agg ccc aag gcc agg agc agc agc gcacgc tgg gct ctc acc 150 Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser Ala ArgTrp Ala Leu Thr 25 30 35 tgc tgc ctg gtg ttg ctc ccc ttc ctt gca gga ctcacc aca tac ctg 198 Cys Cys Leu Val Leu Leu Pro Phe Leu Ala Gly Leu ThrThr Tyr Leu 40 45 50 ctt gtc agc cag ctc cgg gcc cag gga gag gcc tgt gtgcag ttc cag 246 Leu Val Ser Gln Leu Arg Ala Gln Gly Glu Ala Cys Val GlnPhe Gln 55 60 65 70 gct cta aaa gga cag gag ttt gca cct tca cat cag caagtt tat gca 294 Ala Leu Lys Gly Gln Glu Phe Ala Pro Ser His Gln Gln ValTyr Ala 75 80 85 cct ctt aga gca gac gga gat aag cca agg gca cac ctg acagtt gtg 342 Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg Ala His Leu Thr ValVal 90 95 100 aga caa act ccc aca cag cac ttt aaa aat cag ttc cca gctctg cac 390 Arg Gln Thr Pro Thr Gln His Phe Lys Asn Gln Phe Pro Ala LeuHis 105 110 115 tgg gaa cat gaa cta ggc ctg gcc ttc acc aag aac cga atgaac tat 438 Trp Glu His Glu Leu Gly Leu Ala Phe Thr Lys Asn Arg Met AsnTyr 120 125 130 acc aac aaa ttc ctg ctg atc cca gag tcg gga gac tac ttcatt tac 486 Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser Gly Asp Tyr Phe IleTyr 135 140 145 150 tcc cag gtc aca ttc cgt ggg atg acc tct gag tgc agtgaa atc aga 534 Ser Gln Val Thr Phe Arg Gly Met Thr Ser Glu Cys Ser GluIle Arg 155 160 165 cga gca ggc cga cca aac aag cca gac tcc atc act gtggtc atc acc 582 Arg Ala Gly Arg Pro Asn Lys Pro Asp Ser Ile Thr Val ValIle Thr 170 175 180 aag gta aca gac agc tac cct gag cca acc cag ctc ctcatg ggg acc 630 Lys Val Thr Asp Ser Tyr Pro Glu Pro Thr Gln Leu Leu MetGly Thr 185 190 195 aag tct gta tgc gaa gta ggt agc aac tgg ttc cag cccatc tac ctc 678 Lys Ser Val Cys Glu Val Gly Ser Asn Trp Phe Gln Pro IleTyr Leu 200 205 210 gga gcc atg ttc tcc ttg caa gaa ggg gac aag cta atggtg aac gtc 726 Gly Ala Met Phe Ser Leu Gln Glu Gly Asp Lys Leu Met ValAsn Val 215 220 225 230 agt gac atc tct ttg gtg gat tac aca aaa gaa gataaa acc ttc ttt 774 Ser Asp Ile Ser Leu Val Asp Tyr Thr Lys Glu Asp LysThr Phe Phe 235 240 245 gga gcc ttc tta cta tagtaggtcg aggccggcaaggccggatcc 819 Gly Ala Phe Leu Leu 250 4 251 PRT Homo sapiens 4 Met AlaGlu Asp Leu Gly Leu Ser Phe Gly Glu Thr Ala Ser Val Glu 1 5 10 15 MetLeu Pro Glu His Gly Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser 20 25 30 AlaArg Trp Ala Leu Thr Cys Cys Leu Val Leu Leu Pro Phe Leu Ala 35 40 45 GlyLeu Thr Thr Tyr Leu Leu Val Ser Gln Leu Arg Ala Gln Gly Glu 50 55 60 AlaCys Val Gln Phe Gln Ala Leu Lys Gly Gln Glu Phe Ala Pro Ser 65 70 75 80His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg 85 90 95Ala His Leu Thr Val Val Arg Gln Thr Pro Thr Gln His Phe Lys Asn 100 105110 Gln Phe Pro Ala Leu His Trp Glu His Glu Leu Gly Leu Ala Phe Thr 115120 125 Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser130 135 140 Gly Asp Tyr Phe Ile Tyr Ser Gln Val Thr Phe Arg Gly Met ThrSer 145 150 155 160 Glu Cys Ser Glu Ile Arg Arg Ala Gly Arg Pro Asn LysPro Asp Ser 165 170 175 Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser TyrPro Glu Pro Thr 180 185 190 Gln Leu Leu Met Gly Thr Lys Ser Val Cys GluVal Gly Ser Asn Trp 195 200 205 Phe Gln Pro Ile Tyr Leu Gly Ala Met PheSer Leu Gln Glu Gly Asp 210 215 220 Lys Leu Met Val Asn Val Ser Asp IleSer Leu Val Asp Tyr Thr Lys 225 230 235 240 Glu Asp Lys Thr Phe Phe GlyAla Phe Leu Leu 245 250 5 69 PRT Homo sapiens 5 Glu Lys Lys Glu Leu ArgLys Val Ala His Leu Thr Gly Lys Ser Asn 1 5 10 15 Ser Arg Ser Met ProLeu Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu 20 25 30 Leu Ser Gly Val LysTyr Lys Lys Gly Gly Leu Val Ile Asn Glu Thr 35 40 45 Gly Leu Tyr Phe ValTyr Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys 50 55 60 Asn Asn Leu Pro Leu65 6 66 PRT Mouse 6 Glu Lys Lys Glu Pro Arg Ser Val Ala His Leu Thr GlyAsn Pro His 1 5 10 15 Ser Arg Ser Ile Pro Leu Glu Trp Glu Asp Thr TyrGly Thr Ala Leu 20 25 30 Ile Ser Gly Val Lys Tyr Lys Lys Gly Gly Leu ValIle Asn Glu Thr 35 40 45 Phe Val Tyr Ser Lys Val Tyr Phe Arg Gly Gln SerCys Asn Asn Gln 50 55 60 Pro Leu 65 7 66 PRT Rat 7 Glu Thr Lys Lys ProArg Ser Val Ala His Leu Thr Gly Asn Pro Arg 1 5 10 15 Ser Arg Ser IlePro Leu Glu Trp Glu Asp Thr Tyr Gly Thr Ala Leu 20 25 30 Ile Ser Gly ValLys Tyr Lys Lys Gly Gly Leu Val Ile Asn Glu Ala 35 40 45 Phe Val Tyr SerLys Val Tyr Phe Arg Gly Gln Ser Cys Asn Ser Gln 50 55 60 Pro Leu 65 8 71PRT Homo sapiens 8 Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile SerGlu Ala Ser 1 5 10 15 Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu LysGly Tyr Tyr Thr 20 25 30 Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly LysGln Leu Thr Val 35 40 45 Lys Arg Gln Tyr Ile Tyr Ala Gln Val Thr Phe CysSer Asn Arg Glu 50 55 60 Ala Ser Ser Gln Ala Pro Phe 65 70 9 74 PRTMouse 9 Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val Ser Glu Ala Asn1 5 10 15 Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys Gly Tyr TyrThr 20 25 30 Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys Gln Leu ThrVal 35 40 45 Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln Val Thr Phe CysSer 50 55 60 Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe 65 70 10 77 PRTMouse 10 Gly Lys Pro Glu Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Ala1 5 10 15 Ser Ile Pro Ser Gly Ser His Lys Val Thr Leu Ser Ser Trp TyrHis 20 25 30 Asp Arg Gly Trp Ala Lys Ile Ser Asn Met Thr Leu Ser Asn GlyLys 35 40 45 Leu Arg Val Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn IleCys 50 55 60 Phe Arg His His Glu Thr Ser Gly Ser Val Pro Thr Asp 65 7075 11 77 PRT Homo sapiens 11 Ser Lys Leu Glu Ala Gln Pro Phe Ala His LeuThr Ile Asn Ala Thr 1 5 10 15 Asp Ile Pro Ser Gly Ser His Lys Val SerLeu Ser Ser Trp Tyr His 20 25 30 Asp Arg Gly Trp Ala Lys Ile Ser Asn MetThr Phe Ser Asn Gly Lys 35 40 45 Leu Ile Val Asn Gln Asp Gly Phe Tyr TyrLeu Tyr Ala Asn Ile Cys 50 55 60 Phe Arg His His Glu Thr Ser Gly Asp LeuAla Thr Glu 65 70 75 12 85 PRT Homo sapiens 12 Glu Arg Gly Pro Gln ArgVal Ala Ala His Ile Thr Gly Thr Arg Gly 1 5 10 15 Arg Ser Asn Thr LeuSer Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu 20 25 30 Gly Arg Lys Ile AsnSer Trp Glu Ser Ser Arg Ser Gly His Ser Phe 35 40 45 Leu Ser Asn Leu HisLeu Arg Asn Gly Glu Leu Val Ile His Glu Lys 50 55 60 Gly Phe Tyr Tyr IleTyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu 65 70 75 80 Ile Lys Glu AsnThr 85 13 87 PRT Mouse 13 Gly Gly Arg Pro Gln Lys Val Ala Ala His IleThr Gly Ile Thr Arg 1 5 10 15 Arg Ser Asn Ser Ala Leu Ile Pro Ile SerLys Asp Gly Lys Thr Leu 20 25 30 Gly Gln Lys Ile Glu Ser Trp Glu Ser SerArg Lys Gly His Ser Phe 35 40 45 Leu Asn His Val Leu Phe Arg Asn Gly GluLeu Val Ile Glu Gln Glu 50 55 60 Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg PheGln Glu Ala Glu Asp Ala 65 70 75 80 Ser Lys Met Val Ser Lys Asp 85 14 64PRT Homo sapiens 14 Arg Ala Pro Phe Lys Lys Ser Trp Ala Tyr Leu Gln ValAla Lys His 1 5 10 15 Leu Asn Lys Thr Lys Leu Ser Trp Asn Lys Asp GlyIle Leu His Gly 20 25 30 Val Arg Tyr Gln Asp Gly Asn Leu Val Ile Gln PhePro Phe Ile Ile 35 40 45 Cys Gln Leu Gln Phe Leu Val Gln Cys Pro Asn AsnSer Val Asp Leu 50 55 60 15 64 PRT Mouse 15 Ser Thr Pro Ser Lys Lys SerTrp Ala Tyr Leu Gln Val Ser Lys His 1 5 10 15 Leu Asn Asn Thr Lys LeuSer Trp Asn Glu Asp Gly Thr Ile His Gly 20 25 30 Leu Ile Tyr Gln Asp GlyAsn Leu Ile Val Gln Phe Pro Phe Ile Val 35 40 45 Cys Gln Leu Gln Phe LeuVal Gln Cys Ser Asn His Ser Val Asp Leu 50 55 60 16 73 PRT Homo sapiens16 Asp Leu Ser Pro Gly Leu Pro Ala Ala His Leu Ile Gly Ala Pro Leu 1 510 15 Lys Gly Gln Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala Phe Leu 2025 30 Thr Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro Gln 3540 45 Asp Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg Ala Pro Pro Gly 5055 60 Gly Gly Asp Pro Gln Gly Arg Ser Val 65 70 17 75 PRT Mouse 17 AspLeu Asn Pro Glu Leu Pro Ala Ala His Leu Ile Gly Ala Trp Met 1 5 10 15Ser Gly Gln Gly Leu Ser Trp Glu Ala Ser Gln Glu Glu Ala Phe Leu 20 25 30Arg Ser Gly Ala Gln Phe Ser Pro Thr His Gly Leu Ala Leu Pro Gln 35 40 45Asp Gly Val Tyr Tyr Leu Tyr Cys His Val Gly Tyr Arg Gly Arg Thr 50 55 60Pro Pro Ala Gly Arg Ser Arg Ala Arg Ser Leu 65 70 75 18 75 PRT Homosapiens 18 Ala His Ser Thr Leu Lys Pro Ala Ala His Leu Ile Gly Asp ProSer 1 5 10 15 Lys Gln Asn Ser Leu Leu Trp Arg Ala Asn Thr Asp Arg AlaPhe Leu 20 25 30 Gln Asp Gly Phe Ser Leu Ser Asn Asn Ser Leu Leu Val ProThr Ser 35 40 45 Gly Ile Tyr Phe Val Tyr Ser Gln Val Val Phe Ser Gly LysAla Tyr 50 55 60 Ser Pro Lys Ala Thr Ser Ser Pro Leu Tyr Leu 65 70 75 1972 PRT Mouse 19 Thr His Gly Ile Leu Lys Pro Ala Ala His Leu Val Gly TyrPro Ser 1 5 10 15 Lys Gln Asn Ser Leu Leu Trp Arg Ala Ser Thr Asp ArgAla Phe Leu 20 25 30 Arg His Gly Phe Ser Leu Ser Asn Asn Ser Leu Leu IlePro Thr Ser 35 40 45 Phe Val Tyr Ser Gln Val Val Phe Ser Gly Glu Ser CysSer Pro Arg 50 55 60 Ala Ile Pro Thr Pro Ile Tyr Leu 65 70 20 71 PRTHomo sapiens 20 Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala AsnPro Gln 1 5 10 15 Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala AsnAla Leu Leu 20 25 30 Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val ValPro Ser Glu 35 40 45 Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys GlyGln Gly Cys 50 55 60 Pro Ser Thr His Val Leu Leu 65 70 21 70 PRT Mouse21 Gln Asn Ser Ser Asp Lys Pro Val Ala His Val Val Ala Asn His Gln 1 510 15 Val Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg Ala Asn Ala Leu Leu 2025 30 Ala Asn Gly Met Asp Leu Lys Asp Asn Gln Leu Val Val Pro Ala Asp 3540 45 Gly Leu Tyr Leu Val Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys 5055 60 Pro Asp Tyr Val Leu Leu 65 70 22 11 PRT Artificial SequenceDescription of Artificial Sequence Peptide from the HIV TAT protein 22Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10

In the claims:
 1. A method for identifying a compound that modulates thebinding of a first polypeptide to a second polypeptide, the methodcomprising: (a) incubating the first and second polypeptides in thepresence or absence of a test agent, wherein the first polypeptidecomprises an amino acid sequence selected from the group consisting of:(i) the amino acid sequence set forth in SEQ ID NO: 4 with 1 to 25conservative amino acid substitutions, wherein the first polypeptide iscapable of binding the second polypeptide; (ii) the amino acid sequenceset forth in SEQ ID NO: 4 with 1 to 25 amino acid insertions, whereinthe first polypeptide is capable of binding the second polypeptide;(iii) the amino acid sequence set forth in SEQ ID NO: 4 with 1 to 25amino acid deletions, wherein the first polypeptide is capable ofbinding the second polypeptide; (iv) the amino acid sequence set forthin SEQ ID NO: 4 which has a C- and/or N-terminal truncation of 1 to 100amino acid residues, wherein the first polypeptide is capable of bindingthe second polypeptide; and (v) the amino acid sequence set forth in SEQID NO: 4, with from 1 to 25 modifications selected from the groupconsisting of amino acid substitutions, amino acid insertions, aminoacid deletions, C-terminal truncation, and N-terminal truncation,wherein the first polypeptide is capable of binding the secondpolypeptide; wherein the second polypeptide comprises an amino acidsequence selected from the group consisting of (i) the amino acidsequence of NTR3 with 1 to 25 conservative amino acid substitutions,wherein the first polypeptide is capable of binding the secondpolypeptide; (ii) the amino acid sequence of NTR3 with 1 to 25 aminoacid insertions, wherein the first polypeptide is capable of binding thesecond polypeptide; (iii) the amino acid sequence set of NTR3 with 1 to25 amino acid deletions, wherein the first polypeptide is capable ofbinding the second polypeptide; (iv) the amino acid sequence of NTR3which has a C- and/or N-terminal truncation of 1 to 100 amino acidresidues, wherein the first polypeptide is capable of binding the secondpolypeptide; and (v) the amino acid sequence of NTR3, with from 1 to 25modifications selected from the group consisting of amino acidsubstitutions, amino acid insertions, amino acid deletions, C-terminaltruncation, and N-terminal truncation, wherein the first polypeptide iscapable of binding the second polypeptide; and (b) measuring bindingbetween the first and second polypeptides in the presence and absence ofthe test agent, wherein differential binding in the presence versus theabsence of the test agent identifies the test agent as a compound thatmodulates the binding.
 2. The method of claim 1, wherein at least one ofthe first and second polypeptides is detectably-labeled.
 3. The methodof claim 2, further comprising a washing step prior to the measuringstep to remove at least one of unbound first polypeptide and unboundsecond polypeptide.
 4. The method of claim 1, wherein at least one ofthe first and second polypeptides is attached to a substrate.
 5. Themethod of claim 1, wherein the first polypeptide is expressed on thesurface of cells.
 6. The method of claim 1, wherein the firstpolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4.7. The method of claim 1, wherein the second polypeptide comprises theamino acid sequence of NTR3.
 8. The method of claim 1, wherein at leastone of the first and second polypeptides is fused to a heterologouspeptide.
 9. The method of claim 8, wherein the heterologous peptide is aFc fragment.
 10. The method according to claim 1 further comprising astep of making a composition comprising the compound identified in step(b) as a modulator and a pharmaceutically acceptable carrier.
 11. Themethod of claim 1, wherein the test agent is an antibody.
 12. The methodof claim 1, wherein the test agent is a polypeptide comprising anantigen binding fragment of an antibody that specifically binds thefirst or second polypeptide.
 13. The method of claim 1, wherein the testagent is a monoclonal antibody.
 14. The method of claim 1, wherein thetest agent is a humanized antibody.
 15. The method of claim 1, whereinthe test agent is a fully human antibody.
 16. The method of claim 1,wherein the test agent is an antibody that binds to the firstpolypeptide.
 17. The method of claim 1, wherein the test agent is anantibody that binds to the second polypeptide.
 18. The method of claim1, wherein the test agent is a fragment of the first polypeptide thatbinds the second polypeptide.
 19. The method of claim 1, wherein thetest agent is a fragment of the second polypeptide that binds the firstpolypeptide.
 20. The method according to claim 1 for identifying anantagonist of the binding between the first polypeptide and the secondpolypeptide, comprising selecting as an antagonist a test agent thatreduces the binding between the first polypeptide and the secondpolypeptide.
 21. The method according to claim 1 or for identifying anagonist of the binding between the first polypeptide and the secondpolypeptide, comprising selecting as an agonist a test agent thatincreases the binding between the first polypeptide and the secondpolypeptide.
 22. A method for identifying a compound that modulates thebinding of a first polypeptide to a second antibody, the methodcomprising: (a) incubating the first and second polypeptides in thepresence or absence of a test agent, wherein the first polypeptidecomprises an amino acid sequence at least 90% identical to anextracellular domain comprising amino acid residues 57 to 251 of SEQ IDNO: 4, wherein the second polypeptide comprises an amino acid sequenceat least 90% identical to the amino acid sequence of NTR3; and (b)measuring binding between the first and second polypeptides in thepresence and absence of the test agent, wherein differential binding inthe presence versus the absence of the test agent identifies the testagent as a compound that modulates the binding.
 23. The method accordingto claim 22, wherein the first polypeptide comprises amino acid residues57 to 251 of SEQ ID NO:
 4. 24. The method of claim 22, wherein at leastone of the first and second polypeptides is fused to a heterologouspeptide.
 25. The method of claim 24, wherein the heterologous peptide isa Fc fragment.
 26. A method for identifying a compound that modulatesthe binding of a first polypeptide to a second polypeptide, the methodcomprising: (a) incubating the first and second polypeptides in thepresence or absence of a test agent, wherein the second polypeptidecomprises an amino acid sequence at least 90% identical to the aminoacid sequence of NTR3, wherein the first polypeptide comprises an aminoacid sequence selected from the group consisting of: (i) the amino acidsequence set forth in SEQ ID NO: 4 with 1 to 25 conservative amino acidsubstitutions, wherein the first polypeptide is capable of binding thesecond polypeptide; (ii) the amino acid sequence set forth in SEQ ID NO:4 with 1 to 25 amino acid insertions, wherein the first polypeptide iscapable of binding the second polypeptide; (iii) the amino acid sequenceset forth in SEQ ID NO: 4 with 1 to 25 amino acid deletions, wherein thefirst polypeptide is capable of binding the second polypeptide; (iv) theamino acid sequence set forth in SEQ ID NO: 4 which has a C- and/orN-terminal truncation of 1 to 100 amino acid residues, wherein the firstpolypeptide is capable of binding the second polypeptide; and (v) theamino acid sequence set forth in SEQ ID NO: 4, with from 1 to 25modifications selected from the group consisting of amino acidsubstitutions, amino acid insertions, amino acid deletions, C-terminaltruncation, and N-terminal truncation, wherein the first polypeptide iscapable of binding the second polypeptide; and (b) measuring bindingbetween the first and second polypeptides in the presence and absence ofthe test agent, wherein differential binding in the presence versus theabsence of the test agent identifies the test agent as a compound thatmodulates the binding.
 27. A method for identifying a compound thatmodulates the binding of a first polypeptide to a second polypeptide,the method comprising: (a) incubating the first and second polypeptidesin the presence or absence of a test agent; providing the firstpolypeptide comprises an amino acid sequence at least 90% identical tothe amino acid sequence set forth in SEQ ID NO: 4, wherein the secondpolypeptide comprises an amino acid sequence selected from the groupconsisting of (i) the amino acid sequence of NTR3 with 1 to 25conservative amino acid substitutions, wherein the first polypeptide iscapable of binding the second polypeptide; (ii) the amino acid sequenceof NTR3 with 1 to 25 amino acid insertions, wherein the firstpolypeptide is capable of binding the second polypeptide; (iii) theamino acid sequence of NTR3 with 1 to 25 amino acid deletions, whereinthe first polypeptide is capable of binding the second polypeptide; (iv)the amino acid sequence of NTR3 which has a C- and/or N-terminaltruncation of 1 to 100 amino acid residues, wherein the firstpolypeptide is capable of binding the second polypeptide; and (v) theamino acid sequence of NTR3, with from 1 to 25 modifications selectedfrom the group consisting of amino acid substitutions, amino acidinsertions, amino acid deletions, C-terminal truncation, and N-terminaltruncation, wherein the first polypeptide is capable of binding thesecond polypeptide; and (b) measuring binding between the first andsecond polypeptides in the presence and absence of the test agent,wherein differential binding in the presence versus the absence of thetest agent identifies the test agent as a compound that modulates thebinding.
 28. A method for identifying a compound that modulates thebinding of a first polypeptide to a second polypeptide, the methodcomprising: (a) incubating the first and second polypeptides in thepresence or absence of a test agent, wherein the first polypeptidecomprises an amino acid sequence at least 90% identical to the aminoacid sequence set forth in SEQ ID NO: 4, wherein the second polypeptidecomprises an amino acid sequence at least 90% identical to the aminoacid sequence of NTR3; and (b) measuring binding between the first andsecond polypeptides in the presence and absence of the test agent,wherein differential binding in the presence versus the absence of thetest agent identifies the test agent as a compound that modulates thebinding.